Methods for methylation enrichment using preferential ligation of adapters

Abstract

The disclosure relates to enrichment and analysis of methylated DNA through preferential ligation of adapters to methylated, and optionally to partially methylated, DNA. Specifically, the disclosure provides methods of preferentially ligating adapters to methylated DNA in a sample by contacting DNA with a methyl-sensitive deaminase, thereby providing a converted DNA sample in which least a portion of unmethylated CpGs in the DNA are converted to UpGs. A first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. One or more adapters is preferentially ligated to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample, and the adapter-ligated DNA can be sequenced to assess, e.g., methylation status.

Description

Attorney Docket No. GH0197WO METHODS FOR METHYLATION ENRICHMENT USING PREFERENTIAL LIGATION OF ADAPTERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 570,986, filed March 28, 2024, US Provisional Patent Application No.63 / 667,401, filed July 3, 2024, US Provisional Patent Application No.63 / 669,078, filed July 9, 2024, US Provisional Patent Application No.63 / 669,082, filed July 9, 2024, US Provisional Patent Application No. 63 / 670,034, filed July 11, 2024, and US Provisional Patent Application No.63 / 670,035, filed July 11, 2024, each of which is incorporated by reference herein in its entirety for all purposes. FIELD OF THE INVENTION

[0002] The disclosure relates to methods for preferentially ligating adapters to methylated DNA by selective deamination of DNA using a methyl-sensitive deaminase, and preferentially ligating adapters to the methylated DNA of the converted sample. Such methods can be useful for enrichment of methylated DNA and for accurately detecting the methylation status and variants present in DNA, which, in turn, can be used to infer information about the cells and subject from which the DNA sample is derived. In some embodiments, the DNA molecule is from a subject having or suspected of having a disease or disorder, such as cancer. SEQUENCE LISTING

[0003] The present application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “GH0197WO.xml” created on March 27, 2025, which is 191,084 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. INTRODUCTION AND SUMMARY

[0004] Cancer is responsible for millions of deaths per year worldwide. Early cancer detection may result in improved outcomes because early-stage cancer tends to be more susceptible to treatment.

[0005] Improperly controlled cell growth is a hallmark of cancer. Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some resulting in improperly regulated cell division. Such mutations commonly include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations,Attorney Docket No. GH0197WO and inversions. Cancers may also exhibit an accumulation of epigenetic changes, including modification of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors. Thus, cancer can be indicated by non-sequence modifications, such as methylation. Examples of methylation changes in cancer include local gains of DNA methylation, e.g., in the CpG islands at the transcription start sites of genes involved in normal growth control, DNA repair, cell cycle regulation, and / or cell differentiation. Hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. Furthermore, without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. The DNA from such cells may differ epigenetically from shed DNA in a healthy subject. As such, the distribution of epigenetically modified (e.g., methylated) DNA in certain DNA samples, such as cell-free DNA (cfDNA), may change upon carcinogenesis. Thus, sufficiently sensitive epigenetic (e.g., DNA methylation) profiling can be used to detect aberrant methylation in DNA of a sample.

[0006] Biopsies represent a traditional approach for detecting or diagnosing cancer in which cells or tissue are extracted from a possible cancer site and analyzed for relevant phenotypic and / or genotypic features. Biopsies have the drawback of being invasive. Cancer detection based on analysis of body fluids (“liquid biopsies”), such as blood, is an intriguing alternative based on the observation that DNA from cancer cells is released into body fluids. A liquid biopsy is noninvasive (sometimes requiring only a blood draw). However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material in part because the amount of nucleic acids released into body fluids is low and variable, as is recovery of nucleic acids from such fluids in analyzable form. Further, the contribution of DNA from cells in or around a cancer or neoplasm to a sample may be relatively small relative to the contribution from other cells, and the DNA contributed from other cells may be uninformative as to cancer status. Isolating and processing cell-free DNA useful for further analysis in liquid biopsy procedures can be a useful part of these methods.

[0007] Current methods of cancer diagnostic assays of cell-free nucleic acids (e.g., cell-free DNA or cell-free RNA) may focus on the detection of tumor-related somatic variants, including single nucleotide variants (SNVs), copy number variations (CNVs), fusions, and indels (i.e., insertions or deletions), which are all mainstream targets for liquid biopsy. There is growingAttorney Docket No. GH0197WO evidence that non-sequence modifications like methylation status and fragmentomic signal in cell-free DNA can provide information on the source of cell-free DNA and disease level. Detailed knowledge of the non-sequence modifications of the cell-free DNA (e.g., when combined with somatic mutation calling) can improve assessments of tumor status.

[0008] Accordingly, there is a continued need for improved methods and compositions for analyzing non-sequence modifications (such as methylation status) in DNA, including cell-free DNA, e.g., in liquid biopsies.

[0009] The present disclosure aims to meet the need for improved analysis of methylated DNA, such as in a cfDNA sample, provide other benefits, or at least provide the public with a useful choice. In some embodiments, the present disclosure provides methods for preferentially ligating adapters to methylated DNA in a sample through selective deamination of DNA using a methyl- sensitive deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated cytosines and is double-stranded. In some embodiments, one or more adapters are preferentially ligated to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the disclosed methods can improve enrichment and analysis of methylated DNA in a sample by contacting DNA in the sample with a methyl-sensitive deaminase, and preferentially ligating one or more adapters to a portion of the converted DNA sample that comprises unconverted methylated CpGs and is double-stranded.

[0010] Accordingly, the embodiments described herein are provided, which include, but are not limited to, the following.

[0011] Embodiment 1 is a method of preferentially ligating adapters to methylated DNA in a sample, the method comprising: (a) contacting DNA in the sample with a methyl-sensitive deaminase, thereby providing a converted DNA sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs, wherein DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded;Attorney Docket No. GH0197WO (b) preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample.

[0012] Embodiment 2 is the method of the immediately preceding embodiment, further comprising, prior to step (b) and after step (a): subjecting the DNA of the converted DNA sample to end repair to generate end-repaired DNA molecules.

[0013] Embodiment 3 is the method of the immediately preceding embodiment, wherein the end repair is performed using a uracil-tolerant DNA polymerase.

[0014] Embodiment 4 is the method of the immediately preceding embodiment, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially-single stranded DNA during step (a), and wherein the DNA of the second and third portions are preferentially end repaired relative to the DNA of the first portion.

[0015] Embodiment 5 is the method of the immediately preceding embodiment, wherein the uracil-tolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0016] Embodiment 6 is the method of any one of embodiments 3-5, wherein the uracil-tolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0017] Embodiment 7 is the method of embodiment 2, wherein the end repair is performed using a uracil-intolerant DNA polymerase.

[0018] Embodiment 8 is the method of the immediately preceding embodiment, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a), wherein the DNA of the second portion is preferentially end repaired relative to the DNA of the first and third portions.

[0019] Embodiment 9 is the method of any one of embodiments 7-8, wherein the uracil- intolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0020] Embodiment 10 is the method of any one of embodiments 7-8, wherein the uracil- intolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.Attorney Docket No. GH0197WO

[0021] Embodiment 11 is the method of embodiment 1, further comprising, prior to step (a): subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules.

[0022] Embodiment 12 is the method of the immediately preceding embodiment, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a), wherein the one or more adapters are preferentially ligated to the second portion of the DNA is relative to the DNA of the first and third portions.

[0023] Embodiment 13 is the method of embodiment 11 or 12, wherein the end repair is performed using a DNA polymerase that has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0024] Embodiment 14 is the method of embodiment 11 or 12, wherein the end repair is performed using a DNA polymerase that does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0025] Embodiment 15 is the method of any one of embodiments 2-14, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs)in which at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0026] Embodiment 16 is the method of the immediately preceding embodiment, wherein the at least one type of dNTP comprises a modified base, wherein the modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).

[0027] Embodiment 17 is the method of embodiment 1, further comprising, prior to step (b) and after step (a): digesting at least a portion of the DNA of the first portion of the converted DNA sample using a single-stranded DNA (ssDNA) nuclease, thereby providing a digested DNA sample comprising double-stranded DNA.

[0028] Embodiment 18 is the method of the immediately preceding embodiment, wherein the converted DNA sample further comprises a third portion comprising DNA comprising bothAttorney Docket No. GH0197WO converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially double-stranded DNA during step (a).

[0029] Embodiment 19 is the method of the immediately preceding embodiment, wherein, prior to step (b) and after step (a), at least a portion of the DNA of the third portion of the converted DNA is digested using the ssDNA nuclease to produce double-stranded DNA.

[0030] Embodiment 20 is the method of any one of embodiments 17-19, wherein the ssDNA nuclease comprises an ssDNA endonuclease, an ssDNA exonuclease with 5’ to 3’ activity, an ssDNA exonuclease with 3’ to 5’ activity, or any combination thereof. Embodiment 20.1 is the method of any one of embodiments 17-20, wherein the ssDNA nuclease comprises an ssDNA endonuclease. Embodiment 20.2 is the method of any one of embodiments 17-20.1, wherein the ssDNA nuclease comprises an ssDNA exonuclease with 5’ to 3’ activity. Embodiment 20.3 is the method of any one of embodiments 17-20.2, wherein the ssDNA nuclease comprises an ssDNA exonuclease with 3’ to 5’ activity. Embodiment 20.4 is the method of any one of embodiments 17-20.1, wherein the ssDNA nuclease comprises an ssDNA exonuclease with 3’ to 5’ activity and an ssDNA exonuclease with 3’ to 5’ activity.

[0031] Embodiment 21 is the method of any one of embodiments 17-20.4, wherein the ssDNA endonuclease comprises mung bean nuclease, nuclease P1, or nuclease S1. Embodiment 21.1 is the method of the immediately preceding embodiment, wherein the ssDNA endonuclease comprises mung bean nuclease. Embodiment 21.2 is the method of embodiment 21 or 21.1, wherein the ssDNA endonuclease comprises nuclease P1. Embodiment 21.3 is the method of any one of embodiments 21-21.2, wherein the ssDNA endonuclease comprises nuclease S1.

[0032] Embodiment 22 is the method of any one of embodiments 17-21.3, wherein the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease I, exonuclease T, or exonuclease VII. Embodiment 22.1 is the method of embodiment 22, wherein the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease I. Embodiment 22.2 is the method of embodiment 22 or 22.1, wherein the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease T.Attorney Docket No. GH0197WO Embodiment 22.3 is the method of any one of embodiments 22-22.2, wherein the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease VII.

[0033] Embodiment 23 is the method of any one of embodiments 17-22.3, wherein the ssDNA exonuclease with 5’ to 3’ activity comprises exonuclease VII or RecJf. Embodiment 23.1 is the method of embodiment 23, wherein the ssDNA exonuclease with 5’ to 3’ activity comprises RecJf. Embodiment 23.2 is the method of embodiment 23 or 23.1, wherein the ssDNA exonuclease with 5’ to 3’ activity comprises exonuclease VII.

[0034] Embodiment 24 is the method of any one of the preceding embodiments, wherein denaturing comprises heating the converted DNA sample.

[0035] Embodiment 25 is the method of the immediately preceding embodiment, wherein the heating the converted DNA sample comprises heating to a temperature of about 70 degrees Celsius to about 100 degrees Celsius.

[0036] Embodiment 26 is the method of any one of the preceding embodiments, further comprising contacting the converted DNA sample with a single-stranded DNA binding protein (SSB).

[0037] Embodiment 27 is the method of any one of the preceding embodiments, further comprising contacting the DNA of the first portion, DNA of the third portion, or DNA of the first portion and DNA of the third portion with a single-stranded DNA binding protein (SSB).

[0038] Embodiment 28 is the method of any one of embodiments 2-27, further comprising performing an A-tailing reaction, optionally after (a) a step of subjecting the converted DNA sample to end repair, (b) a step of subjecting the DNA sample to end repair, or (c) a step of digesting at least a portion of the first portion of the converted DNA sample using a ssDNA nuclease.

[0039] Embodiment 29 is the method of the immediately preceding embodiment, wherein the end-repair and the A-tailing reactions are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reactions are performed a single tube and / or optionally wherein the end-repair and the A-tailing reactions are performed without an intervening clean-up step.

[0040] Embodiment 30 is the method of any one of embodiments 28-29, wherein the A-tailing reaction is performed using a DNA polymerase that does not possess 5’-3’ exonuclease activityAttorney Docket No. GH0197WO and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

[0041] Embodiment 31 is the method of any one of embodiments 28-30, wherein the A-tailing is performed using a thermostable DNA polymerase.

[0042] Embodiment 32 is the method of any one of the preceding embodiments, wherein the ligating uses a T4 DNA ligase or T3 DNA ligase.

[0043] Embodiment 33 is the method of any one of the preceding embodiments, wherein the one or more adapters comprise molecular barcodes.

[0044] Embodiment 34 is the method of any one of the preceding embodiments, wherein the one or more adapters are Y-shaped adapters.

[0045] Embodiment 35 is the method of any one of the preceding embodiments, further comprising amplifying the converted DNA sample or the digested DNA sample using a DNA polymerase, optionally wherein the amplifying the converted DNA sample or the digested DNA sample is performed after step (b).

[0046] Embodiment 36 is the method of the immediately preceding embodiment, wherein the DNA polymerase is a uracil-intolerant DNA polymerase.

[0047] Embodiment 37 is the method of embodiment 35, wherein the DNA polymerase is a uracil-tolerant DNA polymerase.

[0048] Embodiment 38 is the method of any one of the preceding embodiments, wherein the methyl-sensitive deaminase is thermally inactivated after step (a).

[0049] Embodiment 39 is the method of any one of the preceding embodiments, wherein the methyl-sensitive deaminase is a dsDNA deaminase.

[0050] Embodiment 40 is the method of any one of the preceding embodiments, wherein the methyl-sensitive deaminase is a CpG-specific deaminase.

[0051] Embodiment 41 is the method of the any one of the preceding embodiments, wherein the methyl-sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.

[0052] Embodiment 42 is the method of any one of the preceding embodiments, further comprising, prior to step (a), performing a methylation-preserving amplification of the DNA of the sample.

[0053] Embodiment 43 is the method of the immediately preceding embodiment, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification.Attorney Docket No. GH0197WO

[0054] Embodiment 44 is the method of any one of embodiments 42-43, wherein the methylation-preserving amplification comprises contacting the DNA with a methyltransferase.

[0055] Embodiment 45 is the method of any one of embodiments 42-44, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

[0056] Embodiment 46 is the method of any one of embodiments 42-44, wherein the methylation-preserving amplification comprises thermocycled amplification.

[0057] Embodiment 47 is the method of any one of embodiments 42-44, wherein the methylation-preserving amplification comprises isothermal amplification.

[0058] Embodiment 48 is the method of any one of the preceding embodiments, wherein the step of contacting the DNA in the sample with a methyl-sensitive deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs, comprises single-enzyme 5-methylcytosine sequencing (SEM-seq).

[0059] Embodiment 49 is the method of any one of the preceding embodiments, further comprising sequencing at least a portion of the converted DNA sample or at least a portion of the digested DNA sample, optionally wherein the sequencing the converted DNA sample or the digested DNA sample is performed after step (b), further optionally wherein the sequencing is performed after amplifying the converted DNA sample or the digested DNA sample after step (b).

[0060] Embodiment 50 is the method of any one of the preceding embodiments, further comprising quantifying a level of methylation at one or more differentially methylated regions of the converted DNA sample or at one or more differentially methylated regions of the digested DNA sample, optionally wherein the quantifying the level of methylation of the converted DNA sample or the digested DNA sample is performed after step (b), further optionally wherein the quantifying the level of methylation is performed after amplifying the converted DNA sample or the digested DNA sample after step (b).

[0061] Embodiment 51 is the method of the immediately preceding embodiment, wherein quantifying the level of methylation at one or more differentially methylated regions of the converted DNA sample or the digested DNA sample comprises sequencing at least a portion of the amplified DNA.Attorney Docket No. GH0197WO

[0062] Embodiment 52 is the method of any one of embodiments 49-51, wherein the sequencing comprises next-generation sequencing (NGS).

[0063] Embodiment 53 is the method of the immediately preceding embodiment, wherein the NGS comprises pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization.

[0064] Embodiment 54 is the method of any one of embodiments 49-51, wherein the sequencing comprises single-molecule real time (SMRT) sequencing.

[0065] Embodiment 55 is the method of any one of embodiments 49-51, wherein the sequencing comprises long-read sequencing.

[0066] Embodiment 56 is the method of any one of embodiments 49-51, wherein the sequencing comprises nanopore-based sequencing.

[0067] Embodiment 57 is the method of any one of embodiments 49-51, wherein the sequencing comprises 5-letter or 6-letter sequencing.

[0068] Embodiment 58 is the method of embodiment 49-51 or 56, wherein the sequencing comprises nanopore-based sequencing and the method further comprises subjecting the converted DNA sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base including a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, dUTP, a dNTP comprising fluorodeoxyuridine (FldU), a dNTP comprising 5-iododeoxyuridine (IdU), and / or a dNTP comprising 5- ethynyldeoxyuridine (EdU), and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0069] Embodiment 59 is the method of embodiment 49-51 or 54, wherein the sequencing comprises single-molecule real time (SMRT) sequencing and the method further comprises subjecting the converted DNA sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base including a dNTP comprising a 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, and / or a dNTP comprising 8oxoG, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0070] Embodiment 60 is the method of any one of embodiments 49-59, further comprising analyzing at least some of the sequence data corresponding to regions that are not identified asAttorney Docket No. GH0197WO being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the DNA sample.

[0071] Embodiment 61 is the method of any one of embodiments 49-60, wherein the method further comprises detecting the methylation status of cytosines in the DNA molecules, and wherein the analyzing the sequence data further comprises filtering out the one or more repaired regions of the end-repaired DNA such that the one or more repaired regions are not used to determine the methylation status of cytosines in the DNA sample.

[0072] Embodiment 62 is the method of any one of embodiments 49-60, wherein the method is for detecting the single nucleotide variants (SNVs) in the DNA sample, and wherein the analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support.

[0073] Embodiment 63 is the method of any one of embodiments 49-62, wherein the DNA sample comprises cell-free DNA (cfDNA).

[0074] Embodiment 64 is the method of the immediately preceding embodiment, further comprising analyzing the sequence data to determine a level of measured artifacts in the cfDNA.

[0075] Embodiment 65 is the method of any one of embodiments 2-64, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is other than 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0076] Embodiment 66 is the method of any one of embodiments 2-64, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0077] Embodiment 67 is the method of any one of the preceding embodiments, wherein the DNA in the sample comprises barcodes.

[0078] Embodiment 68 is the method of any one of the preceding embodiments, further comprising enriching the DNA for a plurality of target regions.

[0079] Embodiment 69 is the method of the immediately preceding embodiment, wherein the enriching the DNA occurs prior to a step of amplifying converted DNA, prior to a step ofAttorney Docket No. GH0197WO sequencing the DNA, and / or after contacting the DNA in the sample with the methyl-sensitive deaminase.

[0080] Embodiment 70 is the method of embodiment 68 or 69, wherein the plurality of target regions comprises epigenetic target regions.

[0081] Embodiment 71 is the method of embodiment 70, wherein the epigenetic target regions comprise hypermethylation variable target regions.

[0082] Embodiment 72 is the method of embodiment 70 or 71, wherein the epigenetic target regions comprise hypomethylation variable target regions.

[0083] Embodiment 73 is the method of any one of embodiments 68-72, wherein the plurality of target regions comprise sequence-variable target regions.

[0084] Embodiment 74 is the method of any one of the preceding embodiments, wherein the sample comprises DNA from a formalin fixed paraffin embedded sample.

[0085] Embodiment 75 is the method of any one of embodiments 1-73, wherein the sample comprises cell-free DNA (cfDNA).

[0086] Embodiment 76 is the method of any one of the preceding embodiments, wherein the sample is a blood sample and / or a tissue sample.

[0087] Embodiment 77 is the method of the immediately preceding embodiment, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

[0088] Embodiment 78 is the method of any one of the preceding embodiments, wherein the sample is from a subject.

[0089] Embodiment 79 is the method of any one of the preceding embodiments, wherein the sample is from a subject and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.

[0090] Embodiment 80 is the method of any one of embodiments 78-79, wherein the subject is an animal.

[0091] Embodiment 81 is the method of the immediately preceding embodiment, wherein the subject is a human.

[0092] Embodiment 82 is the method of any one of embodiments 78-81, wherein the subject has or is at risk of having a cancer.

[0093] Embodiment 83 is the method of any one of embodiments 78-82, further comprising determining the presence or status of a cancer in the subject.Attorney Docket No. GH0197WO

[0094] In some embodiments, the results of the methods disclosed herein are used as an input to generate a report. The report may be in a paper or electronic format. For example, the true methylation status of cytosines or variants, as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such a report. Alternatively or additionally, diagnostic information or therapeutic recommendations which are at least in part based on the methods disclosed herein can be included in the report.

[0095] The various steps of the methods disclosed herein may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and / or by the same or different people.

[0096] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG.1 is a schematic showing an exemplary methylation enrichment method wherein adapters are preferentially ligated to methylated DNA. Deamination of unmethylated CpGs in the DNA using a methyl-sensitive deaminase is performed on a DNA sample, which results in denaturation of substantially unmethylated strands or portions thereof (indicated with white arrows), followed by end-repair using a uracil-tolerant polymerase and A-tailing reactions, preferential ligation of next-generation sequencing (NGS) adapters to double-stranded DNA comprising unconverted methylated CpGs relative to single-stranded, denatured DNA comprising converted UpGs, uracil-tolerant amplification of the DNA, optional enrichment, sequencing, and methylation calling. G to A substitutions at CpG sites indicates that a sequence was resynthesized during end repair where the opposite strand contained an unmethylated cytosine that was deaminated to U before templating incorporation of a U during end repair. The sequences in the figure are: mCGmCGmCGmCGmCG (SEQ ID NO: 90), mCGmCGmCGCGCG (SEQ ID NO: 91), CGCGmCGmCGmCG (SEQ ID NO: 92), mCGmCGCGCGCG (SEQ ID NO: 96), CGCGCGmCGmCG (SEQ ID NO: 97), mCGCGCGCGCG (SEQ ID NO: 98), CGCGCGCGmCG (SEQ ID NO: 99), CGCGCGCGCG (SEQ ID NO: 93), mCGmCGmCGUGUG (SEQ ID NO: 109), UGUGmCGmCGmCG (SEQ ID NO: 110), mCGmCGUGUGUG (SEQ ID NO: 111), UGUGUGmCGmCG (SEQ ID NO: 112), mCGUGUGUGUG (SEQ ID NO: 113), UGUGUGUGmCG (SEQ ID NO: 114),Attorney Docket No. GH0197WO UGUGUGUGUG (SEQ ID NO: 108), mCGmCGmCGmCAmCA (SEQ ID NO: 115), and mCGmCGmCAmCAmCA (SEQ ID NO: 116), wherein “mC” denotes a methylated cytosine.

[0098] FIG.2 is a schematic showing an exemplary methylation enrichment method wherein adapters are preferentially ligated to methylated DNA. Deamination of unmethylated CpGs in the DNA using a methyl-sensitive deaminase is performed on a DNA sample, which results in denaturation of substantially unmethylated strands or portions thereof (indicated with white arrows), followed by end-repair using a uracil-intolerant polymerase and A-tailing reactions, ligation of NGS adapters preferentially to double-stranded DNA comprising unconverted methylated CpGs relative to single-stranded, denatured DNA comprising converted UpGs, amplification (e.g., uracil-intolerant amplification) of the double-stranded DNA, optional enrichment, sequencing, and methylation calling. Using a uracil-intolerant polymerase results in incomplete resynthesis opposite strands that underwent deamination and thus contain uracils. The incomplete resynthesis can prevent adapter ligation, thus facilitating preferential ligation of adapters to highly methylated, fully double-stranded DNA. The sequences in the figure are: mCGmCGmCGmCGmCG (SEQ ID NO: 90), mCGmCGmCGCGCG (SEQ ID NO: 91), CGCGmCGmCGmCG (SEQ ID NO: 92), mCGmCGCGCGCG (SEQ ID NO: 96), CGCGCGmCGmCG (SEQ ID NO: 97), mCGCGCGCGCG (SEQ ID NO: 98), CGCGCGCGmCG (SEQ ID NO: 99), CGCGCGCGCG (SEQ ID NO: 93), mCGmCGmCGUGUG (SEQ ID NO: 109), UGUGmCGmCGmCG (SEQ ID NO: 110), mCGmCGUGUGUG (SEQ ID NO: 111), UGUGUGmCGmCG (SEQ ID NO: 112), mCGUGUGUGUG (SEQ ID NO: 113), UGUGUGUGmCG (SEQ ID NO: 114), and UGUGUGUGUG (SEQ ID NO: 108), wherein “mC” denotes a methylated cytosine.

[0099] FIG.3 is a schematic showing an exemplary methylation enrichment method wherein adapters are preferentially ligated to methylated DNA. End-repair and A-tailing reactions are performed on a DNA sample, followed by deamination of unmethylated CpGs in the DNA using a methyl-sensitive deaminase, which results in denaturation of substantially unmethylated strands or portions thereof (indicated with white arrows), ligation of NGS adapters preferentially to double-stranded DNA comprising unconverted methylated CpGs relative to single-stranded, denatured DNA comprising converted UpGs, uracil-intolerant or uracil-tolerant amplification of the double-stranded DNA, optional enrichment, sequencing, and methylation calling. Denaturation of unmethylated DNA via deamination facilitates preferential ligation of adapters to methylated DNA. The sequences in the figure are: mCGmCGmCGmCGmCG (SEQ ID NO:Attorney Docket No. GH0197WO 90), mCGmCGmCGCGCG (SEQ ID NO: 91), CGCGmCGmCGmCG (SEQ ID NO: 92), mCGmCGCGCGCG (SEQ ID NO: 96), CGCGCGmCGmCG (SEQ ID NO: 97), mCGCGCGCGCG (SEQ ID NO: 98), CGCGCGCGmCG (SEQ ID NO: 99), CGCGCGCGCG (SEQ ID NO: 93), mCGmCGmCGUGUG (SEQ ID NO: 109), UGUGmCGmCGmCG (SEQ ID NO: 110), mCGmCGUGUGUG (SEQ ID NO: 111), UGUGUGmCGmCG (SEQ ID NO: 112), mCGUGUGUGUG (SEQ ID NO: 113), UGUGUGUGmCG (SEQ ID NO: 114), and UGUGUGUGUG (SEQ ID NO: 108), wherein “mC” denotes a methylated cytosine.

[0100] FIG.4 is a schematic showing an exemplary methylation enrichment method wherein adapters are preferentially ligated to methylated DNA. Deamination of unmethylated CpGs in the DNA using a methyl-sensitive deaminase is performed on a DNA sample, which results in denaturation of substantially unmethylated strands or portions thereof (indicated with white arrows), followed by single-stranded DNA nuclease treatment, ligation of NGS adapters preferentially to double-stranded DNA comprising unconverted methylated CpGs relative to digested DNA comprising converted UpGs, uracil-tolerant or uracil-intolerant amplification of the double-stranded DNA, optional enrichment, sequencing, and methylation calling. Denaturation of unmethylated DNA via deamination facilitates preferential ligation of adapters to methylated DNA. Adapter ligation can be a blunt-ended ligation, or an A-tailing step can be performed after nuclease treatment and before adapter ligation. Single-stranded DNA nuclease treatment degrades unmethylated DNA that was deaminated and denatured, thereby facilitating preferential ligation of adapters to methylated DNA. The sequences in the figure are: mCGmCGmCGmCGmCG (SEQ ID NO: 90), mCGmCGmCGCGCG (SEQ ID NO: 91), CGCGmCGmCGmCG (SEQ ID NO: 92), mCGmCGCGCGCG (SEQ ID NO: 96), CGCGCGmCGmCG (SEQ ID NO: 97), mCGCGCGCGCG (SEQ ID NO: 98), CGCGCGCGmCG (SEQ ID NO: 99), CGCGCGCGCG (SEQ ID NO: 93), mCGmCGmCGUGUG (SEQ ID NO: 109), UGUGmCGmCGmCG (SEQ ID NO: 110), mCGmCGUGUGUG (SEQ ID NO: 111), UGUGUGmCGmCG (SEQ ID NO: 112), mCGmCGUGUGmCGmCG (SEQ ID NO: 117), UGUGUGUGmCG (SEQ ID NO: 114), and UGUGUGUGUG (SEQ ID NO: 108), wherein “mC” denotes a methylated cytosine.

[0101] FIG.5 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure. DETAILED DESCRIPTIONAttorney Docket No. GH0197WO

[0102] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosure will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the disclosure as defined by the appended claims.

[0103] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids.

[0104] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0105] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components.

[0106] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way.

[0107] All patents, patent applications, websites, other publications or documents and the like cited herein whether supra or infra, are expressly incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated. I. Definitions

[0108] A “reaction cleanup” refers to the removal of contaminants such as salts, enzymes, unincorporated dNTPs, primers, ethidium bromide, and other impurities that can interfere with downstream analysis. For example, when a reaction cleanup is performed between end repair and an A-tailing reaction, it removes unincorporated dNTPs such that the A-tailing reaction can be performed solely in the presence of dATP (i.e. not dCTP, dGTP and dCTP, as used in the end tailing reaction). Reaction cleanups can be performed using commercially available kits such as MinElute Reaction Cleanup Kit (Qiagen)Attorney Docket No. GH0197WO

[0109] “Repaired regions”, also referred to as “synthesized regions” or “regions of the end-repaired DNA that were synthesized during the end repair” refer to regions of the DNA that were not present in the DNA prior to the end repair and A-tailing reactions. They are regions which have been synthesized by the polymerases used in the end repair and / or A tailing reactions, if present. In instances where the A-tailing is performed in the same tube as the end repair reaction, all four types of dNTPs will be present, and thus the polymerases used for A- tailing may generate synthesized regions, e.g. through nick translation. In instances where the A- tailing is performed separately to the end repair reaction, and these steps are separated by a reaction cleanup, only dATP will be present in the A-tailing reaction, and thus the polymerases used for A-tailing will not typically generate synthesized regions because the dNTP components are not all present in the A-tailing reaction mix.

[0110] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair with G, for example, is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0111] A “type of dNTP” refers to a dNTP comprising a specific base, including A, T, G or C. Accordingly, wherein an end repair reaction is performed with dNTPs, wherein at least one type of dNTP comprises a modified base, the end repair reaction may be performed using dCTP comprising 5mC, and dATP, dTTP and dGTP all comprising non-modified bases.

[0112] “Capable of identifying the base modification in the at least one type of dNTP” refers to the ability of a modification-sensitive sequencing method to detect the presence or absence of the base modification in the at least one type of dNTP comprising a modified base used in the end repair. This detection of the base modification may be direct, such as in nanopore sequencing or single molecule real time sequencing, wherein the sequencing data itself indicates the presence or absence of a base modification. Alternatively, the detection of the base modification may be indirect, for example wherein the method involves a conversion procedure which alters the base pairing specificity dependent on the base modification status. It is these changes in base pairing specificity which can be detected by the sequencing method, e.g. through the comparison of the sequencing data to a reference sequence. Moreover, a modification-Attorney Docket No. GH0197WO sensitive sequencing method is capable of identifying the base modification in the at least one type of dNTP regardless of whether it can distinguish one base modification from all other base modifications. For example, one form of modification-sensitive sequencing is sequencing after bisulfite conversion. This method is capable of distinguishing 5hmC and 5mC from unmethylated cytosine, but cannot distinguish 5hmC from 5mC.

[0113] Bases of the “same identity” refer to the same base, regardless of modification status of that base. For example, cytosine is considered to be the “same identity” as 5- methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.

[0114] “Cell-free DNA,” “cfDNA molecules,” or simply “cfDNA” include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) into a fluid found in the organism, and may be obtained from a sample of the fluid without the need to perform an in vitro cell lysis step.

[0115] As used herein, “cellular nucleic acids” means nucleic acids that are disposed within one or more cells from which the nucleic acids have originated, at least at the point a sample is taken or collected from a subject, even if those nucleic acids are subsequently removed (e.g., via cell lysis) as part of a given analytical process.

[0116] DNA is “derived from cancerous cells” if it originated from a tumor cell. Cell free DNA derived from cancerous cells includes ctDNA or circulating tumor DNA. Tumor cells are neoplastic cells that originated from a tumor, regardless of whether they remain in the tumor or become separated from the tumor (as in the cases, e.g., of metastatic cancer cells and circulating tumor cells).

[0117] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ ^ 3’ direction of the nucleic acid sequence)). In some embodiments, DNA methylation refers to addition of a methyl group to adenine, such as in N6- methyladenine (6mA). In some embodiments, DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring). In some embodiments, 5-methylation refers to addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosineAttorney Docket No. GH0197WO (5mC). In some embodiments, methylation comprises a derivative of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon in the 3rdposition of the cytosine ring). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0118] The “modified nucleoside profile of DNA” means the position and identity of the nucleoside and the modification status of the nucleoside, such as methylations, within a DNA sequence. As described above, different modification sensitive sequencing methods can be used to detect such modifications. This includes methods which involve conversion followed by sequencing detect one or more different types of modified or unmodified nucleoside. For example, the TAPS method detects, but does not distinguish between, 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosine (5hmC). Hence, a method for analyzing the modified nucleoside profile of DNA in a sample typically means identifying particular modifications or groups of modification, such as 5mC and / or 5hmC. Modified nucleosides are identified according to the specific method / conversion procedure being used as described above. This generally involves comparing sequence data obtained from DNA that has been subjected to a conversion procedure to a reference sequence. Typically, the method involves (i) comparing the sequence data with (A) one or more pre-determined reference sequence; or (B) sequence data obtained by sequencing a sub-sample of the DNA that was not subjected to the conversion procedure, for example a subsample that was separated before subjecting a separate subsample to the conversion procedure, for example as described herein; and (ii) identifying point differences between the converted DNA sequences and the reference sequence(s) (A) or non-converted DNA sequences (B) as nucleosides (in the initial sample) having a modification status that permits a change in base pairing specificity on exposure to the conversion procedure.Attorney Docket No. GH0197WO

[0119] As used herein, a modification or other feature is present in “a greater proportion” in a first sample or population of nucleic acid than in a second sample or population when the fraction of nucleotides with the modification or other feature is higher in the first sample or population than in the second population. For example, if in a first sample, one tenth of the nucleotides are mC, and in a second sample, one twentieth of the nucleotides are mC, then the first sample comprises the cytosine modification of 5-methylation in a greater proportion than the second sample.

[0120] As used herein, “without substantially altering base-pairing specificity” of a given nucleobase means that a majority of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample. In some embodiments, 75%, 90%, 95%, or 99% of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample.

[0121] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair with G is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0122] As used herein, “modified cytosine” refers to a cytosine in which at least one position of the cytosine has been substituted with a chemical moiety, such as a methyl or hydroxymethyl, that is different from the substituent at that position in unmodified cytosine. For the avoidance of doubt, “modified cytosine” does not include unmodified cytosine.

[0123] As used herein, a “combination” comprising a plurality of members refers to either of a single composition comprising the members or a set of compositions in proximity, e.g., in separate containers or compartments within a larger container, such as a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0124] The “capture yield” of a collection of probes for a given target set refers to the amount (e.g., amount relative to another target set or an absolute amount) of nucleic acidAttorney Docket No. GH0197WO corresponding to the target set that the collection of probes captures under typical conditions. Exemplary typical capture conditions are an incubation of the sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (about 20 µL) containing stringent hybridization buffer. The capture yield may be expressed in absolute terms or, for a plurality of collections of probes, relative terms. When capture yields for a plurality of sets of target regions are compared, they are normalized for the footprint size of the target region set (e.g., on a per- kilobase basis). Thus, for example, if the footprint sizes of first and second target regions are 50 kb and 500 kb, respectively (giving a normalization factor of 0.1), then the DNA corresponding to the first target region set is captured with a higher yield than DNA corresponding to the second target region set when the mass per volume concentration of the captured DNA corresponding to the first target region set is more than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set. As a further example, using the same footprint sizes, if the captured DNA corresponding to the first target region set has a mass per volume concentration of 0.2 times the mass per volume concentration of the captured DNA corresponding to the second target region set, then the DNA corresponding to the first target region set was captured with a two-fold greater capture yield than the DNA corresponding to the second target region set.

[0125] “Capturing” one or more target nucleic acids refers to preferentially isolating or separating the one or more target nucleic acids from non-target nucleic acids.

[0126] A “captured set” of nucleic acids refers to nucleic acids that have undergone capture.

[0127] A “target-region set” or “set of target regions” refers to a plurality of genomic loci targeted for capture and / or targeted by a set of probes (e.g., through sequence complementarity).

[0128] “Corresponding to a target region set” means that a nucleic acid, such as cfDNA, originated from a locus in the target region set or specifically binds one or more probes for the target-region set.

[0129] As used herein, a “differentially methylated region” (DMR) refers to a region of DNA having a detectably different degree of methylation in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type; or having a detectably different degree of methylation in at least one cell or tissue type obtained from a subject having a disease or disorder relative to the degree of methylation in the same region of DNA in the same cell or tissue type obtained from a healthy subject. In someAttorney Docket No. GH0197WO embodiments, a DMR has a detectably higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject. In some embodiments, a DMR has a detectably lower degree of methylation (e.g., a hypomethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject.

[0130] “Specifically binds” in the context of a probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or replicates thereof, to form a stable probe:target hybrid, while at the same time formation of stable probe:non-target hybrids is minimized. Thus, a probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non- target sequence, to enable capture or detection of the target sequence. Appropriate hybridization conditions are well-known in the art, may be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51, 11.12- 11.13, 11.45-11.47 and 11.55-11.57, incorporated by reference herein).

[0131] “Sequence-variable target region set” refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).

[0132] “Epigenetic target region set” refers to a set of target regions that may show sequence-independent changes in neoplastic cells (e.g., tumor cells and cancer cells) or that may show sequence-independent changes in cfDNA from subjects having cancer relative to cfDNA from healthy subjects. Examples of sequence-independent changes include, but not limited to, changes in methylation (increases or decreases), nucleosome distribution, CTCF binding, transcription start sites, and regulatory protein binding regions. For present purposes, loci susceptible to neoplasia-, tumor-, or cancer-associated focal amplifications and / or gene fusions may also be included in an epigenetic target region set because detection of a change in copy number by sequencing or a fused sequence that maps to more than one locus in a reference genome tends to be more similar to detection of exemplary epigenetic changes discussed aboveAttorney Docket No. GH0197WO than detection of nucleotide substitutions, insertions, or deletions, e.g., in that the focal amplifications and / or gene fusions can be detected at a relatively shallow depth of sequencing because their detection does not depend on the accuracy of base calls at one or a few individual positions.

[0133] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypermethylated DNA can include DNA molecules comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, or at least 10 methylated residues.

[0134] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules comprising 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0135] As used herein, “methylation status” can refer to the presence or absence of methyl group on a DNA base (e.g. cytosine) at a particular genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., highly methylated, low methylated, intermediately methylated or unmethylated nucleic acid molecules). The methylation status can also refer to the number of nucleotides methylated in a particular nucleic acid molecule.

[0136] As used herein, “mutation” refers to a variation from a known reference sequence and includes mutations such as, for example, single nucleotide variants (SNVs), and insertions or deletions (indels). A mutation can be a germline or somatic mutation. In some embodiments, a reference sequence for purposes of comparison is a wildtype genomic sequence of the species of the subject providing a test sample, typically the human genome.

[0137] As used herein, the terms “neoplasm” and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subject. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor is referred to as a cancer or a cancerous tumor.Attorney Docket No. GH0197WO

[0138] As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next-generation sequencing includes the use of instruments capable of sequencing single molecules. Examples of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0139] As used herein, “nucleic acid tag” refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length), used to distinguish nucleic acids from different samples (e.g., representing a sample index), distinguish nucleic acids from different partitions (e.g., representing a partition tag) or different nucleic acid molecules in the same sample (e.g., representing a molecular barcode), of different types, or which have undergone different processing. The nucleic acid tag comprises a predetermined, fixed, non-random, random or semi-random oligonucleotide sequence. Such nucleic acid tags may be used to label different nucleic acid molecules or different nucleic acid samples or sub-samples. Nucleic acid tags can be single-stranded, double-stranded, or at least partially double-stranded. Nucleic acid tags optionally have the same length or varied lengths. Nucleic acid tags can also include double-stranded molecules having one or more blunt-ends, include 5’ or 3’ single-stranded regions (e.g., an overhang), and / or include one or more other single-stranded regions at other locations within a given molecule. Nucleic acid tags can be attached to one end or to both ends of the other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information such as the sample of origin, form, or processing of a given nucleic acid. For example, nucleic acid tags can also be used to enable pooling and / or parallel processing of multiple samples comprising nucleic acids bearing different molecular barcodes and / or sample indexes in which the nucleic acids are subsequently being deconvolved by detecting (e.g., reading) the nucleic acid tags. Nucleic acid tags can also be referred to as identifiers (e.g. molecular identifier, sample identifier). Additionally, or alternatively, nucleic acid tags can be used as molecular identifiers (e.g., to distinguish between different molecules or amplicons of different parent molecules in the same sample or sub-sample). This includes, for example, uniquely tagging different nucleic acidAttorney Docket No. GH0197WO molecules in a given sample, or non-uniquely tagging such molecules. In the case of non-unique tagging applications, a limited number of tags (i.e., molecular barcodes) may be used to tag each nucleic acid molecule such that different molecules can be distinguished based on their endogenous sequence information (for example, start and / or stop positions where they map to a selected reference genome, a sub-sequence of one or both ends of a sequence, and / or length of a sequence) in combination with at least one molecular barcode. Typically, a sufficient number of different molecular barcodes are used such that there is a low probability (e.g., less than about a 10%, less than about a 5%, less than about a 1%, or less than about a 0.1% chance) that any two molecules may have the same endogenous sequence information (e.g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode. Terms such as “library adaptors having distinct molecular barcodes” encompass library adaptors for uniquely or non-uniquely tagging molecules, in that regardless of whether the adaptors are for unique or non-unique tagging, distinct barcodes will be present in the population of adaptors.

[0140] As used herein, DNA that is “not immobilized” or that is “free in solution” refers to DNA that is not bound covalently or non-covalently to a solid support, such as a bead. Such DNA may be free in solution during any step (such as all steps) of the disclosed methods.

[0141] As used herein, “partitioning” refers to physically separating or fractionating a mixture of nucleic acid molecules in a sample based on a characteristic of the nucleic acid molecules. The partitioning can be physical partitioning of molecules. Partitioning can involve separating the nucleic acid molecules into groups or sets based on the level of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned based on the level of methylation of the nucleic acid molecules. In some embodiments, the methods and systems used for partitioning may be found in PCT Patent Application No. PCT / US2017 / 068329, which is hereby incorporated by reference in its entirety.

[0142] As used herein, “partitioned set” or “partition” refers to a set of nucleic acid molecules partitioned into a set or group based on the differential binding affinity of the nucleic acid molecules or proteins associated with the nucleic acid molecules to a binding agent. A partitioned set may also be referred to as a subsample. The binding agent binds preferentially to the nucleic acid molecules comprising nucleotides with epigenetic modification. For example, if the epigenetic modification is methylation, the binding agent can be a methyl binding domain (MBD) protein. In some embodiments, a partitioned set can comprise nucleic acid moleculesAttorney Docket No. GH0197WO belonging to a particular level or degree of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned into three sets – one set for highly methylated nucleic acid molecules (first subsample, hyper partition, hyper partitioned set or hypermethylated partitioned set), a second set for low methylated nucleic acid molecules (second subsample, hypo partition, hypo partitioned set or hypomethylated partitioned set), and a third set for intermediate methylated nucleic acid molecules (third subsample, intermediate partitioned set, intermediately methylated partitioned set, residual partitioned set, or residual partition). In another example, the nucleic acid molecules can be partitioned based on the number of methylated nucleotides - one partitioned set can have nucleic acid molecules with nine methylated nucleotides, and another partitioned set can have unmethylated nucleic acid molecules (zero methylated nucleotides).

[0143] The terms “agent that recognizes a modified nucleobase in DNA,” such as an “agent that recognizes a modified cytosine in DNA” refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, such as methyl cytosine. A “modified nucleobase” is a nucleobase that comprises a difference in chemical structure from an unmodified nucleobase. In the case of DNA, an unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, a modified nucleobase is a modified cytosine. In some embodiments, a modified nucleobase is a methylated nucleobase. In some embodiments, a modified cytosine is a methyl cytosine, e.g., a 5-methyl cytosine. In such embodiments, the cytosine modification is a methyl. Agents that recognize a methyl cytosine in DNA include but are not limited to “methyl binding reagents,” which refer herein to reagents that bind to a methyl cytosine. Methyl binding reagents include but are not limited to methyl binding domains (MBDs) and methyl binding proteins (MBPs). In some such embodiments, the DNA may be single- stranded or double-stranded. Suitable agents include agents that recognize modified nucleotides in double-stranded DNA, single-stranded DNA, and both double-stranded and single-stranded DNA.

[0144] As used herein, “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG”, the nucleotides are in 5’ ^ 3’ orderAttorney Docket No. GH0197WO from left to right, and in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases.

[0145] As used herein, “processing” refers to a set of steps used to generate a library of nucleic acids that is suitable for sequencing. The set of steps can include, but are not limited to, partitioning, end repairing, addition of sequencing adapters, tagging, and / or PCR amplification of nucleic acids.

[0146] As used herein, “quantitative measure” refers to an absolute or relative measure. A quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low). A quantitative measure can be a ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure.

[0147] As used herein, “reference sequence” refers to a known sequence used for purposes of comparison with experimentally determined sequences. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. A reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include non-contiguous segments that align with different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as, hg19 and hg38.

[0148] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0149] As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid- phase sequencing, high-throughput sequencing, massively parallel signature sequencing,Attorney Docket No. GH0197WO emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single- molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performed by a gene analyzer such as, for example, gene analyzers commercially available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many others.

[0150] As used herein, “sequence information” in the context of a nucleic acid polymer means the order and identity of monomer units (e.g., nucleotides, etc.) in that polymer.

[0151] As used herein “sequence-variable target region set” refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).

[0152] As used herein, the terms “somatic mutation” or “somatic variation” are used interchangeably. They refer to a mutation in the genome that occurs after conception. Somatic mutations can occur in any cell of the body except germ cells and accordingly, are not passed on to progeny.

[0153] As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject”. For example, a subject can be an individual who has been diagnosed with having a cancer, is going to receive a cancer therapy, and / or has received at least one cancer therapy. The subject can be in remission of a cancer. As another example, the subject can be an individual who is diagnosed of having an autoimmune disease. As another example, the subject can be a female individual who is pregnant or who is planning on getting pregnant, who may have been diagnosed of or suspected of having a disease, e.g., a cancer, an auto-immune disease.Attorney Docket No. GH0197WO

[0154] As used herein, “target-region set” or “set of target regions” or “target regions” or “target regions of interest” or “regions of interest” or “genomic regions of interest” refers to a plurality of genomic loci or a plurality of genomic regions targeted for capture and / or targeted by a set of probes (e.g., through sequence complementarity).

[0155] As used herein, “tumor fraction” refers to the proportion of cfDNA molecules that originated from tumor cells for a given sample, or sample-region pair.

[0156] As used herein, an “asymmetric adapter” is a double stranded adapter in which the two strands are not completely complementary or are otherwise distinguishable such that synthesis of a complementary sequence of one strand of the adapter results in a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y-shaped adapters and bubble adapters.

[0157] As used herein, a “Y-shaped adapter” refers to an adapter comprising two DNA strands comprising complementary and non-complementary parts, wherein the non- complementary parts form single-stranded arms. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that the complementary (double-stranded) part of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded arms may or may not be of identical length.

[0158] As used herein, a “bubble adapter” refers to an adapter comprising two DNA strands comprising a non-complementary part flanked by complementary parts, such that the adapter has a single stranded region located between double-stranded regions. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that one of the complementary (double-stranded) parts of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter that would be attached to the insert or sample molecule may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded portions of the two strands may or may not be of identical length.

[0159] The terms “or a combination thereof” and “or combinations thereof” as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinationsAttorney Docket No. GH0197WO that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0160] “Buffy coat” refers to the portion of a blood (such as whole blood) or bone marrow sample that contains all or most of the white blood cells and platelets of the sample. The buffy coat fraction of a sample can be prepared from the sample using centrifugation, which separates sample components by density. For example, following centrifugation of a whole blood sample, the buffy coat fraction is situated between the plasma and erythrocyte (red blood cell) layers. The buffy coat can contain both mononuclear (e.g., T cells, B cells, NK cells, dendritic cells, and monocytes) and polymorphonuclear (e.g., granulocytes such as neutrophils and eosinophils) white blood cells.

[0161] As used herein, “leukapheresis” refers to a procedure in which white blood cells (leukocytes) are isolated from a sample of blood collected from a subject. Leukapheresis may be performed, e.g., obtain cells for research, diagnostic, prognostic, or monitoring purposes, such as those described herein. Thus, as used herein, a “leukapheresis sample” refers to a sample comprising leukocytes collected from a subject using leukapheresis.

[0162] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and are found in the peripheral circulation. Such cells include, e.g., lymphocytes (T cells, B cells, and NK cells) as well as monocytes, and are isolated from blood samples (such as from a whole blood sample collected from a subject) using density gradient centrifugation.

[0163] As used herein, “amplify,” “amplifying,” or “amplification” refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification methods can include any suitable methods known in the art. As used herein, a nucleic acid molecule amplified using “methylation-preserving amplification” substantially maintains its methylation status post-amplification.

[0164] A “X1nnnX2mutation” in a specified polypeptide as used herein, where X1and X2are amino acids and nnn is a position in an amino acid sequence, refers to a substitution in the polypeptide of amino acid X1present at position nnn of the full-length wild-type polypeptide with amino acid X2. The polypeptide is the human polypeptide unless indicated otherwise. The polypeptide comprising the X1nnnX2mutation may, but does not necessarily, compriseAttorney Docket No. GH0197WO additional differences from the wild-type sequence, including but not limited to truncations and deletions as well as other substitutions. For example, a “T1372S mutation” in TET2 refers to a substitution in a TET2 enzyme of the threonine present at position 1372 of the full-length wild- type human TET2 enzyme with a serine. Position 1372 of wild-type human TET2 aligns to position 258 and 248, respectively, of the truncated TET2 sequences disclosed as SEQ ID NOs: 23 and 24 of US Patent 10,961,525. Similarly, a “V1900X2mutation” where X2is A, C, G, I, or P in TET2 refers to a substitution in a TET2 enzyme of the valine present at position 1900 of the full-length wild-type human TET2 enzyme with an alanine, cysteine, glycine, isoleucine, or proline.

[0165] As used herein, “preferentially ligate” refers to ligation in which there is a greater likelihood that one or more adapters ligate to a first nucleotide sequence relative to a second nucleotide sequence. The differential likelihood that the one or more adapters ligate to the first nucleotide sequence relative to the second nucleotide sequence can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.

[0166] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise. II. Exemplary methods A. Overview

[0167] Many commercialized methods and methods undergoing development target specific changes in methylation, e.g., cancer-related changes that occur in early stage cancers and pre-cancers. However, the methylation detection accuracy, molecular recovery, coverage uniformity, and methylation-specific separation efficacy can be improved in these methods (e.g., single-site methylation assays), which would lead to improved clinical assay performance and / or assay cost reduction.

[0168] Cancer formation and progression may arise from both genetic modification and epigenetic features of deoxyribonucleic acid (DNA). The present disclosure provides methods and systems for preferentially ligating adapters to methylated DNA in a sample, such as cell-free DNA (cfDNA). Preferential adapter ligation to methylated DNA can provide improved clinical assay performance and / or assay cost reduction, e.g., because methylated molecules of interest are more efficiently and / or frequently detected and sequenced, and / or fewer unmethylated moleculesAttorney Docket No. GH0197WO of less interest are detected and sequenced. The present disclosure further provides methods for analyzing epigenetic and / or sequence-variable target regions, e.g., including hypermethylation variable target regions.

[0169] Without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. As such, the distribution of tissue of origin of certain DNA samples, such as cfDNA, may change upon carcinogenesis. Thus, for example, an increase in the level of hypermethylation variable target regions that show lower methylation in healthy cfDNA than in at least one other tissue type can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer. Similarly, an increase in the level of hypomethylation variable target regions in the sample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0170] Thus, DNA methylation profiling can be used to detect aberrant methylation in DNA of a sample. The DNA can correspond to certain genomic regions (“differentially methylated regions” or “DMRs”) that are normally hypermethylated or hypomethylated in a given sample type (e.g., cfDNA from the bloodstream) but which may show an abnormal degree of methylation that correlates to a neoplasm or cancer, e.g., because of unusually increased contributions of tissues to the type of sample (e.g., due to increased shedding of DNA in or around the neoplasm or cancer) and / or from extents of methylation of the genome that are altered during development or that are perturbed by disease, for example, cancer or any cancer- associated disease.

[0171] In some embodiments, DNA methylation comprises addition of a methyl group to a cytosine residue at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ -> 3’ direction of the nucleic acid sequence). In some embodiments, DNA methylation comprises addition of a methyl group to an adenine residue, such as in N6- methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring). In some embodiments, 5-methylation comprises addition of a methyl group to the 5C position of the cytosine residue to create 5-methylcytosine (m5c or 5-mC or 5mC). In some embodiments, methylation comprises a derivative of m5c. Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC or 5hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon in the 3rdposition of the cytosineAttorney Docket No. GH0197WO ring). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine residue to generate 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0172] The present disclosure provides methods and systems for preferentially ligating adapters to methylated DNA, such as cell-free DNA (cfDNA), in a sample. The disclosed methods preferentially ligate adapters to methylated DNA in a sample by contacting the DNA in the sample with a methyl-sensitive deaminase, thereby providing a converted DNA sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. One or more adapters to the DNA are preferentially ligated to the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. Selectively ligating one or more adapters to the DNA molecules can improve the efficiency of downstream analyses, such as library preparation, sequencing, and subsequent data analysis, e.g., analyzing the sequencing data to quantify a level of methylation at one or more differentially methylated regions of the DNA. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during the step of contacting the DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the methylated, double-stranded DNA of the converted DNA sample is amplified, such that resources used downstream, e.g., during sequencing and analysis, can be more efficiently focused on methylated DNA. In some embodiments, the methylated DNA comprises methylated cytosines of CpG dinucleotides.

[0173] Some embodiments of the disclosed methods of preferentially ligating adapters to methylated DNA in a sample comprise: (a) contacting DNA in the sample with a methyl- sensitive deaminase, thereby providing a converted DNA sample in which at least a portion ofAttorney Docket No. GH0197WO unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded; and (b) preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample.

[0174] In some embodiments, the method further comprises, prior to step (b) and after step (a), subjecting the DNA of the converted DNA sample to end repair to generate end-repaired DNA molecules.

[0175] In some embodiments, the end repair is performed using a uracil-tolerant DNA polymerase. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a). In some embodiments, the DNA of the second and third portions are preferentially end repaired relative to the DNA of the first portion. In some embodiments, the uracil-tolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase. In some embodiments, the uracil-tolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0176] In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, OneTaq®DNA Polymerase, Taq DNA Polymerase, LongAmp®Taq DNA Polymerase, Hemo KlenTaq, Epimark®Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3′→5′ exo-), or any combination thereof.

[0177] In some embodiments, the end repair is performed using a uracil-intolerant DNA polymerase. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a). In some embodiments, the DNA of the second portion is preferentially end repaired relative to the DNA of the first and third portions. In some embodiments, one or more adapters areAttorney Docket No. GH0197WO preferentially ligated to the second portion of the DNA relative to the DNA of the first and third portions. In some embodiments, the uracil-intolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase. In some embodiments, the uracil- intolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0178] In some embodiments, the uracil-intolerant DNA polymerase used in the amplification reaction may be Q5® High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Vent® DNA Polymerase, Vent® (exo-) DNA Polymerase, Deep Vent® DNA Polymerase, Deep Vent® (exo-) DNA Polymerase, or any combination thereof. In some embodiments, the uracil-intolerant DNA polymerase used in the amplification reaction may be an Archaeal uracil-intolerant DNA polymerase, such as a uracil-intolerant DNA polymerase from Pyrococcus furiosus or Methanosarcina acetivorans (see, e.g., Wardle J, Burgers PM, Cann IK, Darley K, Heslop P, Johansson E, Lin LJ, McGlynn P, Sanvoisin J, Stith CM, Connolly BA. Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya. Nucleic Acids Res.2008 Feb;36(3):705-11.). In some embodiments, the Archaeal uracil-intolerant DNA polymerase is a Family B DNA polymerase, such as Pfu-Pol or Mac-Pol.

[0179] In some embodiments, the methods further comprise, prior to step (a), subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a). In some embodiments, the one or more adapters are preferentially ligated to the second portion of the DNA is relative to the DNA of the first and third portions. In some embodiments, the end repair is performed using a DNA polymerase that has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase. In some embodiments, the end repair is performed using a DNA polymerase that does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0180] In some embodiments, the end repair is performed using dNTPs in which at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations. In some embodiments, the at least one type of dNTP comprises a modified base. InAttorney Docket No. GH0197WO some embodiments, the modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).

[0181] In some embodiments, the methods further comprise, prior to step (b) and after step (a), digesting at least a portion of the DNA of the first portion of the converted DNA sample using a single-stranded DNA (ssDNA) nuclease, thereby providing a digested DNA sample comprising double-stranded DNA. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially double- stranded DNA during step (a). In some embodiments, prior to step (b) and after step (a), at least a portion of the DNA of the third portion of the converted DNA is digested using the ssDNA nuclease to produce double-stranded DNA.

[0182] In some embodiments, the ssDNA nuclease comprises an ssDNA endonuclease, an ssDNA exonuclease with 5’ to 3’ activity, an ssDNA exonuclease with 3’ to 5’ activity, or any combination thereof. In some embodiments, the ssDNA nuclease comprises an ssDNA endonuclease. In some embodiments, the ssDNA nuclease comprises an ssDNA exonuclease with 5’ to 3’ activity. In some embodiments, the ssDNA nuclease comprises an ssDNA exonuclease with 3’ to 5’ activity. In some embodiments, the ssDNA endonuclease comprises mung bean nuclease, nuclease P1, or nuclease S1. In some embodiments, the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease I, exonuclease T, or exonuclease VII. In some embodiments, the ssDNA exonuclease with 5’ to 3’ activity comprises exonuclease VII or RecJf.

[0183] In some embodiments, the denaturing comprises heating the converted DNA sample. In some embodiments, the heating the converted DNA sample comprises heating to a temperature of about 70 degrees Celsius to about 100 degrees Celsius, about 80 degrees Celsius to about 100 degrees Celsius, about 90 degrees Celsius to about 100 degrees Celsius, or about 88 degrees Celsius, or about 95 degrees Celsius. In some embodiments, the heating occurs for about 1 minute to about 15 minutes, about 2.5 minutes to about 10 minutes, or about 5 minutes. In some embodiments, the method further comprises contacting the converted DNA sample with a single-stranded DNA binding protein (SSB) (e.g., E. coli single single-stranded binding protein, human single-stranded DNA binding protein, or a thermostable single-stranded binding protein).Attorney Docket No. GH0197WO In some embodiments, the binding of the SSB to denatured (i.e., single-stranded DNA), can inhibit the reforming of double-stranded DNA. In some embodiments, the method further comprises comprising contacting the DNA of the first portion, DNA of the third portion, or DNA of the first portion and DNA of the third portion with a single-stranded DNA binding protein (SSB).

[0184] In some embodiments, the methods further comprise performing an A-tailing reaction, optionally after (a) a step of subjecting the converted DNA sample to end repair, (b) a step of subjecting the DNA sample to end repair, or (c) a step of digesting at least a portion of the first portion of the converted DNA sample using a ssDNA nuclease.

[0185] In some embodiments, the methyl-sensitive deaminase is thermally inactivated after contacting the DNA in the sample with the methyl-sensitive deaminase. In some embodiments, the thermal inactivation comprises heating or cooling of the methyl-sensitive deaminase to a temperature at which the methyl-sensitive deaminase has reduced or inhibited activity relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling. In some embodiments, the thermal inactivation completely inhibits the activity of the methyl-sensitive deaminase or reduces the activity of the methyl-sensitive deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling.

[0186] In some embodiments, the methyl-sensitive deaminase is a double-stranded DNA (dsDNA) deaminase. In some embodiments, the dsDNA, methyl-sensitive deaminase has deaminase activity on single-stranded DNA (ssDNA). In some embodiments, the methyl- sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification- sensitive DNA deaminase A (MsddA)-like deaminase. In some embodiments, the methyl- sensitive deaminase is a CpG-specific deaminase.

[0187] The disclosed methods can be combined with analysis of one or more additional biomarkers. In some embodiments, the disclosed methods are combined with one or more methods, such as but not limited to, methods for assessing DNA methylation patterns, DNA mutations (such as somatic mutations), nucleic acid fragmentation patterns, non-coding RNA (such as micro RNAs (miRNAs), ribosomal RNAs, transfer RNAs, small nucleolar RNAs (snow RNAs), and / or small nuclear RNAs (snRNAs)) levels, and / or cell type proportions / levels, cellular locations, and / or structural modifications of one or more proteins (such as in a sampleAttorney Docket No. GH0197WO from a subject). In some embodiments, the disclosed methods are combined with one or more analyses of genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and / or abnormal changes in nucleic acid 5-methylcytosine. B. Samples and Subjects

[0188] The disclosure relates to methods of preferentially ligating adapters to methylated DNA in a sample, comprising contacting the DNA of the sample with a methyl-sensitive deaminase to provide a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. The methods of preferentially ligating adapters to methylated DNA comprises preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some cases, the DNA sample is obtained or has been obtained from a subject. In some embodiments, the DNA sample may comprise or consist of DNA from a biological sample obtained from a subject. The subject may be a human, a mammal, an animal, a primate, rodent (including mice and rats), or other common laboratory, domestic, companion, service or agricultural animal, for example a rabbit, dog, cat, horse, cow, sheep, goat or pig. Preferably, the DNA sample is from a human. The subject may in some cases have or be suspected of having a cancer, tumor, or neoplasm. In other cases, the subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologics. The subject may be in remission, e.g. from a tumor, cancer, or neoplasia (e.g., following treatment such as chemotherapy, surgical resection, radiation, or a combination thereof). The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders. In someAttorney Docket No. GH0197WO embodiments, the sample is a DNA sample obtained from a tumor tissue biopsy. The cancer, tumor, or neoplasm may generally be of any type, for example a cancer tumor or neoplasm of the lung, colon, rectum (or colorectum), kidney, breast, prostate, or liver, or other type of cancer as described herein. In some embodiments, the sample is obtained from a subject in remission from a tumor, cancer, or neoplasia (e.g., following chemotherapy, surgical resection, radiation, or a combination thereof). In any of the foregoing embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia may be of the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the pre- cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the lung. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the breast. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the prostate. In any of the foregoing embodiments, the subject may be a human subject. In some embodiments, the sample is obtained from a subject having a stage I cancer, stage II cancer, stage III cancer or stage IV cancer.

[0189] In some embodiments, the subject may have an infection, a transplant rejection, or other disease or disorder related to changes in the immune system. The subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologics. The subject may be in remission. The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders.

[0190] The biological sample can be any biological sample isolated from a subject. Biological samples can include body tissues, such as known or suspected solid tumors (such as carcinomas, adenocarcinomas, or sarcomas), whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine. In some embodiments, biological samples are body fluids, particularly blood and fractions thereof (e.g., plasma and / or serum), or urine. A sample can be in the form originally isolated from a subject or can have been subjected to furtherAttorney Docket No. GH0197WO processing to remove or add components, such as cells, or enrich for one component relative to another.

[0191] In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject having or suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. The population includes nucleic acids having varying levels of sequence variation and / or epigenetic variation, such as post-translation modifications (PTMs) of chromatin and / or nucleobase modifications, e.g., modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

[0192] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis.

[0193] In a particular embodiment, the DNA sample comprises cell-free DNA. In another particular embodiment the DNA sample is a DNA sample from a formalin fixed paraffin embedded (FFPE) sample.

[0194] In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. The population includes nucleic acids having varying levels of sequence variation, epigenetic variation, and / or post-replication or transcriptional modifications. Post-replication modifications include modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

[0195] In some embodiments, the DNA sample is from plasma. The volume of plasma used to obtain the DNA sample can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume can be 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled plasma may be 5 to 20 mL. In some embodiments, the sample volume is 3-5 mL of plasma, such as 4 mL of plasma, per 10 mL whole blood.

[0196] In some embodiments, the sample comprises whole blood. Exemplary volumes of sampled whole blood are 0.4-40 mL, 5-20 mL, 10-20 mL, 1-6 mL, 1-3 mL, and 3-5 mL. For example, the volume can be 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL,Attorney Docket No. GH0197WO 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled whole blood may be 5 to 20 mL. In some embodiments, the sample volume is 1-5 mL of whole blood, such as 2.5 mL of whole blood.

[0197] In some embodiments, the sample comprises buffy coat separated from whole blood. Exemplary volumes of sampled buffy coat are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled buffy coat may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of buffy coat, such as 0.3 mL of buffy coat, per 10 mL whole blood.

[0198] In some embodiments, the sample comprises PBMCs separated from whole blood. Exemplary volumes of sampled PBMCs are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled PBMCs may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of PBMCs, such as 0.3 mL of PBMCs, per 10 mL whole blood.

[0199] In some embodiments, the sample comprises leukocytes separated from subject blood using leukapheresis. Exemplary volumes of sampled leukocytes from leukapheresis are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, or 20 mL. A volume of sampled leukocytes from leukapheresis may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.6 mL of leukocytes from leukapheresis, such as 0.4 mL of leukocytes, per 10 mL whole blood.

[0200] A sample can comprise various amounts of DNA that contain genome equivalents. For example, a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cell free DNA (cfDNA), about 200 billion (2x1011) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.

[0201] A sample can comprise nucleic acids from different sources, e.g., nucleic acids from cells and cell-free nucleic acids of the same subject, and nucleic acids from cells and cell- free nucleic acids of different subjects. In some embodiments, the nucleic acid may be DNA. A sample can comprise DNA carrying mutations. For example, a sample can comprise DNAAttorney Docket No. GH0197WO carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations existing in germline DNA of a subject. Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., cancer cells. A sample can comprise DNA carrying cancer- associated mutations (e.g., cancer-associated somatic mutations). A sample can comprise an epigenetic variant, wherein the epigenetic variant associated with the presence of a genetic variant such as a cancer-associated mutation. In some embodiments, the sample comprises an epigenetic variant associated with the presence of a genetic variant, wherein the sample does not comprise the genetic variant.

[0202] The DNA sample may be or comprise cell free nucleic acids or cfDNA. The cfDNA may be obtained from a test subject, for example as described above. For example, the sample for analysis may be plasma or serum containing cell-free nucleic acids. “Cell-free DNA” “cfDNA molecules,” or “cfDNA”, for example, include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) in vivo into a fluid found in the organism, and may be obtained by obtaining a sample of the fluid without the need to perform an in vitro cell lysis step. In other words, cell-free nucleic acids or cfDNA are nucleic acids or DNA not contained within or otherwise bound to a cell, or the nucleic acids or DNA remaining in a sample after removing intact cells. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi- interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or a hybrid thereof. A cell-free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluid from cancer cells e.g., circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell free nucleic acids are produced by tumor cells. In some embodiments, cell free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.

[0203] Exemplary amounts of cell-free nucleic acids (e.g. cfDNA) in a sample before amplification range from about 1 fg to about 1 μg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to aboutAttorney Docket No. GH0197WO 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can comprise obtaining 1 femtogram (fg) to 200 ng cell-free nucleic acid molecules from samples.

[0204] Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides.

[0205] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together. Generally, after addition of buffers and wash steps, nucleic acids can be precipitated with an alcohol. Further clean up steps may be used such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, DNA or protein for sequencing, hybridization, and / or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield.

[0206] After such processing, samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA and single stranded RNA. In some embodiments, single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.

[0207] The methods disclosed herein are also particularly suited for the analysis of DNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. While the formalin fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to the DNA within the tissues, such as nicks in the DNA. As explained elsewhere herein, these nicks can lead to synthesis of regions of the DNA molecule in the end repair process. The methods disclosed herein allow for these regions to be identified and the sequence data to be interpreted accordingly.Attorney Docket No. GH0197WO

[0208] Reference or control molecules can be added to or spiked into a sample as a control or normalization standard. For example, a certain amount of modified DNA from a species other than the species of the subject from which the sample was obtained or synthetic nucleic acids comprising certain modifications may be added to the sample. In some embodiments, the reference or control molecules are distinguishable from the molecules originally present in the sample. In some embodiments, the detected DNA sequences are normalized to the reference or control molecules. C. End repair and A-tailing

[0209] In some embodiments, the disclosed methods comprise subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules. In some embodiments, the end repair is performed prior to or after contacting the DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the end repair is performed after contacting the DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the end repair is performed prior to contacting the DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the end repair is performed prior to or after preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the end repair is performed prior to preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the end repair is performed prior to or after contacting the DNA in the sample with a methyl-sensitive deaminase and prior to or after preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the end repair is performed after contacting the DNA in the sample with a methyl-sensitive deaminase and prior to preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the end repair is performed prior to contacting the DNA in the sample with a methyl-sensitive deaminase and prior to preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample.

[0210] In some embodiments, the end repair is performed on the converted DNA sample. In some embodiments, DNA of a first portion of the converted DNA sample comprisesAttorney Docket No. GH0197WO converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs. In some embodiments, the DNA of the third portion is denatured to form partially single- stranded DNA during a step of contacting the DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the DNA of the second and third portions are preferentially end repaired relative to the DNA of the first portion. In some embodiments, the DNA of the second portion is preferentially end repaired relative to the DNA of the first and third portions.

[0211] In some embodiments, the end repair is performed using deoxynucleotide triphosphates (dNTPs). In some embodiments, at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into repaired regions of the end-repaired DNA molecules at one or more locations.

[0212] End repair refers to methods for repairing DNA by the conversion of non-blunt ended DNA into blunt ended DNA. Sequencing workflows typically use end repair to make ends of DNA molecules compatible with adapters, which are subsequently ligated onto the DNA. Fragmented and / or damaged DNA (e.g. cfDNA or DNA from FFPE samples) often contain non- blunt ends, which contain 3’overhangs and / or 5’overhangs. A 3’overhang refers to the 3’ end of a DNA strand which extends beyond the 5’end of the paired strand, resulting in one or more unpaired nucleotides at the 3’end of the DNA strand. Conversely, a 5’overhang refers to the 5’ end of a DNA strand which extends beyond the 3’end of the paired strand, resulting in one or more unpaired nucleotides at the 5’end of the DNA strand.

[0213] The process of end repair involves the conversion of double-stranded DNA with 3’overhangs and / or 5’overhangs to double-stranded DNA without overhangs. This can be done using one or more enzymes such as T4 DNA polymerase and / or Klenow fragment. The 3’ to 5’ exonuclease activity of these enzymes removes the 3’ends at 3’overhangs and the 5’ to 3’ polymerase activity of these enzymes extends the 3’ ends at 5’ overhangs to remove the 5’ overhang, thereby generating a blunt-ended DNA molecule. In order to fill in these 5’ overhangs, end repair is conducted in the presence of dATP, dCTP, dGTP and dTTP. End repair can also include a second step, which involves the addition of a phosphate group to the 5' ends of DNA, by an enzyme such as polynucleotide kinase. This makes the 5’ends of the end-repaired DNA molecules compatible with the subsequent action of DNA polymerases and DNA ligases.Attorney Docket No. GH0197WO

[0214] As used herein, the term “A-tailing” refers to the addition of a single deoxyadenosine residue to the end of a blunt-ended double-stranded DNA fragment to form a 3' deoxyadenosine single-base overhang. Such A tailing reactions are conducted with polymerases which have the ability to add a non-templated A to the 3' end of a blunt, double-stranded DNA molecule. Polymerases capable of A-tailing typically do not possess 3’-5’ exonuclease activity. When A-tailing is performed as a separate reaction to end repair, it is typically conducted in the presence of dATP, but the absence of dCTP, dTTP and dGTP. A-tailed fragments are not compatible for self-ligation (i.e., self-circularizatian and concatenation of the DNA), but they are compatible with 3' T-overhangs, which can be used on adapters. Methods comprising end repair, A-tailing and ligation to adapters with 3' T-overhangs can result in higher efficiency ligation, compared to blunt ended ligation, as blunt ligation can lead to self-ligation of the adapters and / or DNA molecules.

[0215] In some embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by blunt end ligation of adapters. In other embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by A-tailing and sticky-end ligation of T-tailed adapters. In some embodiments, A-tailing can follow a step of subjecting the converted DNA sample to end repair. In some embodiments, A-tailing can follow a step of subjecting the DNA sample to end repair. In some embodiments, A-tailing can follow a step of digesting at least a portion of the first portion of the converted DNA sample using a ssDNA nuclease. When the methods disclosed herein comprise an A-tailing step, it may be performed separately from the end repair with an intervening reaction clean-up step or it may be performed in the same reaction as the end repair (e.g. using NEBNext® Ultra™ II End Repair / dA-Tailing Module (E7546)). In some embodiments, the A-tailing step may be performed in the same reaction as the end repair without an intervening clean-up step. In some embodiments, the reaction clean-up step removes unincorporated dNTPs. In instances wherein the A-tailing reaction is performed in the same reaction as end repair, the sticky-end ligation may be performed with a mixture of T-tailed adapters and C-tailed adapters.

[0216] End repair and A tailing reactions can have varying impacts on the composition of the DNA molecule, dependent on the exact workflow and reaction components used. These reactions can lead to the synthesis of regions at the 3’ends of DNA strands, but also the synthesis of internal regions through nick translation and through gap filling followed by ligation.Attorney Docket No. GH0197WO

[0217] In some embodiments, end repair can lead to 3’fill in with unmethylated cytosines, which may not reflect the true methylation status of that position in the DNA molecule prior to the generation of the 5’overhang. In nicked DNA, polymerases which contain 5’ to 3’ exonuclease activity and / or strand displacement activity can lead to the synthesis of internal regions of the DNA molecule through nick translation. If the end repair reaction is conducted with non-methylated deoxycytidine triphosphate (dCTP), the synthesized regions will incorporate the non-methylated dCTP, potentially at positions which initially comprised methylated cytosines. In gapped DNA, both the DNA polymerases used in end repair and A tailing can lead to the generation of synthesized regions. The gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. After this gap filling, a nick will still exist between the synthesized region and the region of the original DNA molecule 3’ of the gap. The A-tailing enzymes may then introduce further synthesized regions through nick translation, as described for the nicked DNA. This synthesized region may extend to the 3’end of the DNA molecule.

[0218] In some embodiments, the end-repair and the A-tailing reactions are performed in a single tube. In such cases, the A tailing reaction can be performed at a higher temperature than the end repair. Optionally, end repair is performed at ambient temperature (e.g.15-35°C) and A tailing is performed at a temperature over 60°C, including e.g., about 60°C-75°C. The A tailing reaction can be performed using a thermostable polymerase (e.g. Taq DNA polymerase, Tfl DNA polymerase, Bst DNA Polymerase, Large Fragment or Tth DNA polymerase) and the method further comprises increasing temperature of the sample after the end repair to inactivate the polymerase used in end repair (e.g. T4 DNA polymerase or Klenow fragment). In some embodiments the A-tailing is performed using a DNA polymerase that: (i) does not possess 5’-3’ exonuclease activity; and / or (ii) is not a strand displacing DNA polymerase. These properties reduce the ability of the DNA polymerase to extend from nick. This reduces the level of synthesis which may occur during the end repair and A-tailing reactions thus reducing the proportion of sequencing data that may be filtered out as potentially containing artifactual data. Accordingly, in some embodiments, the A-tailing is performed using a DNA polymerase that cannot extend from a nick in the DNA such as HemoKlen Taq. In other embodiments, the A- tailing is performed using Taq DNA polymerase. In other embodiments, the A-tailing is performed using Tfl polymerase, Bst DNA Polymerase, Large Fragment or Tth polymerase.Attorney Docket No. GH0197WO

[0219] . In order to reduce the level of synthesized regions, the end repair reaction can be performed using DNA polymerases can be used which lack 5’to 3’ exonuclease activity and / or strand displacement activity (e.g. T4 DNA polymerase or Klenow fragment).

[0220] In nicked DNA and gapped DNA, nick translation is reduced in end repair through the use of polymerases which lack 5’to 3’ exonuclease activity and / or strand displacement activity. The separation of the end repair and A tailing reaction by a reaction clean- up means that only dATP (not dCTP, dTTP or dGTP) is present in the A tailing reaction. This means that efficient nick translation cannot occur in the A tailing reaction because the three of the four nucleotide components are not present in the reaction mixture. In gapped DNA, the gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. These filled gaps thereby generate synthesized regions.

[0221] In some embodiments, the end-repair is performed with a polymerase which lacks 5’to 3’ exonuclease activity and / or strand displacement activity. In some cases, the polymerase used in the end repair reaction may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Hemo KlenTaq, phi29 DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”) or T4 DNA Polymerase. In some embodiments, the polymerase used in the end repair is T4 DNA Polymerase or Klenow fragment. In some embodiments, the end repair is performed with a DNA polymerase which has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0222] In some embodiments, the end repair is performed with a uracil-tolerant DNA polymerase. In some embodiments, the uracil-tolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase. In some embodiments, the uracil-tolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0223] In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, OneTaq®DNA Polymerase, Taq DNA Polymerase, LongAmp®Taq DNA Polymerase, Hemo KlenTaq, Epimark®Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase IAttorney Docket No. GH0197WO (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3′→5′ exo-), or any combination thereof.

[0224] In some embodiments, the end repair is performed with a uracil-intolerant DNA polymerase. In some embodiments, the uracil-intolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase. In some embodiments, the uracil- intolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0225] In some embodiments, the uracil-intolerant DNA polymerase used in the amplification reaction may be Q5® High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Vent® DNA Polymerase, Vent® (exo-) DNA Polymerase, Deep Vent® DNA Polymerase, Deep Vent® (exo-) DNA Polymerase, or any combination thereof. In some embodiments, the uracil-intolerant DNA polymerase used in the amplification reaction may be an Archaeal uracil-intolerant DNA polymerase, such as a uracil-intolerant DNA polymerase from Pyrococcus furiosus or Methanosarcina acetivorans (see, e.g., Wardle J, Burgers PM, Cann IK, Darley K, Heslop P, Johansson E, Lin LJ, McGlynn P, Sanvoisin J, Stith CM, Connolly BA. Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya. Nucleic Acids Res.2008 Feb;36(3):705-11.). In some embodiments, the Archaeal uracil-intolerant DNA polymerase is a Family B DNA polymerase, such as Pfu-Pol or Mac-Pol.

[0226] In some embodiments, the methods disclosed herein comprise an A tailing reaction after the end repair and before the ligation reaction, wherein the end repair and A tailing reactions are separated by a reaction cleanup. The A tailing reaction is typically performed in the presence of dATP, but in the absence of dCTP, dTTP and dGTP. In some embodiments, the A tailing reaction is performed using Klenow Fragment lacking 3'-5' exonuclease activity.

[0227] In some embodiments, a dNTP that comprises a modified base is used in end repair, which may be any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. In some embodiments, a dNTP comprising a modified base can be used in a combined end repair and A-tailing reaction. In some embodiments, the modified base is incorporated in the synthesized regions at both CpG sites and CpH sites (i.e. CpA, CpC and CpT sites). While methylation of cytosines in non-CpG contexts has been described, it is thought to comprise 0.02% of total methyl-cytosine in differentiated somatic cells (Jang et al. Genes (Basel).2017 Jun; 8(6): 148). The presence of methylated cytosine in non-Attorney Docket No. GH0197WO CpG contexts in the end-repaired DNA can therefore be interpreted as being introduced during the end repair and / or A-tailing reactions. Using a dNTP comprising a modified base can therefore be used to effectively label the synthesized regions of the end repaired DNA.

[0228] In some embodiments, a dNTP that comprises a modified base may comprise any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. The modified base may be 5-caryboxylcytosine (5-caC), 4-methylcytosine (4mC), 5-methylcytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), N6-methyladenosine (6mA), bromodeoxyuridine (BrdU), 5-fluorodeoxyuridine (FldU), 5-iododeoxyuridine (IdU), 5- ethynyldeoxyuridine (EdU) and / or 8-oxoguanine (8oxoG).

[0229] When a dNTP comprising a modified base is used, it may be used in place of the equivalent unmodified base in the end repair reaction. For instance, if a dCTP comprising 5mC is used in the end repair reaction, there may be no dCTP comprising an unmodified cytosine. This would ensure that dCTPs incorporated into the DNA molecule during the end repair reaction contain 5mC. In some embodiments, multiple types of dNTP comprising a modified base are used in the end repair. For example, dATP comprising 6mA and dCTP comprising 5mC can be used in the end repair reaction in place of dATP comprising unmodified adenine and dCTP comprising unmodified cytosine. The use of multiple types of dNTP comprising a modified base is advantageous because it provides increased resolution in defining the regions of the end- repaired DNA molecule which have been synthesized during the end repair reaction. This is because, in this example, the end of a synthesized region can be defined as the first unmodified adenine or unmodified cytosine after a stretch of containing 6mAs and / or 5mCs, rather than relying on the detection of solely an unmodified adenine or solely an unmodified cytosine.

[0230] The sequencing method used will depend on the type of modified base used in the end-repair reaction such that the specific modification can be detected. Exemplary conversion- based methods are described above alongside the base modification which they can detect. Moreover, nanopore-based sequencing can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU, and single-molecule real time (SMRT) sequencing from Pacific Biosciences can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG.

[0231] The disclosed methods use at least one type of dNTP which comprises a modified base (e.g. a methylated deoxycytidine triphosphate, such as deoxycytidine triphosphate comprising 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC)) in the end repair reaction. In particular embodiments, the dNTP is 5mC. During end repair reactions, theAttorney Docket No. GH0197WO methylated deoxycytidine triphosphate will be incorporated into the synthesized regions regardless of the sequence context. This will result in methylated cytosines in non-CpG positions (i.e., methylated cytosines in a CpH context), which are very rare in nature. These methylated non-CpG cytosines can therefore be used as labels for identifying synthesized regions in the end repaired DNA molecule. Similarly, other types of dNTP which comprise a modified base uncommon or absent in nature can be used. The identification of such modified bases can be performed using sequencing, and regions comprising these modifications can be interpreted as defining regions which were synthesized in the end repair reaction.

[0232] In other embodiments of the disclosed methods, the modified base is a methylated cytosine, such as 5mC or 5hmC. In some embodiments, the methylated cytosine is 5mCpH (where H = A, T, or C). In other embodiments, the modified base is other than 5mC or 5hmC. In some embodiments wherein the modified base is other than 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-modified bases spanning a modified base, wherein the bases are of the same identity to the modified bases present in the at least one type of dNTP; and / or (ii) the sequence between a non-modified base and the end of a sequence read, wherein there is no additional non-modified bases between the non-modified base and the end of the sequence read, where the non-modified bases are the same identity as the modified base present in the at least one type of dNTP. In some embodiments wherein the modified base is a methylated cytosine, such as 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-methylated cytosines which span one or more methylated CpH cytosines; and / or (ii) the sequence between a methylated CpH cytosine and an end of a sequence read, wherein the methylated CpH cytosine is the CpH cytosine most distant from the end of the sequence read, or a subsequence thereof comprising one or more methylated CpH cytosines. D. Ligation to Adapters

[0233] In some embodiments, the methods comprise ligating adapters to DNA, which can be performed preferentially with respect to methylated DNA. In some embodiments, DNA molecules can be subjected to blunt-end ligation with blunt-ended adapters. In some embodiments, DNA molecules can be subjected to sticky-end ligation with sticky-ended adapters. In some embodiments, once the DNA has been end-repaired it can be subjected to blunt-end ligation with blunt-ended adapters, in cases where A-tailing is not performed, or sticky end ligation with T-tailed adapters, when A tailing is performed. DNA molecules can be ligated to adapters at either one end or both ends. DNA molecules can be ligated with at least partiallyAttorney Docket No. GH0197WO double stranded adapter (e.g., a Y shaped or bell-shaped adapter). In some embodiments, the ligation step can take place before or after the conversion step. In general, “conversion step” or “conversion procedure” refers to any step or procedure that changes the base pairing specificity of one or more nucleotides. In some embodiments, the conversion step comprises contacting DNA (e.g., DNA in a sample or end-repaired DNA) with a methyl-sensitive deaminase. In some embodiments, the conversion step of contacting DNA (e.g., DNA in a sample or end-repaired DNA) with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. In some embodiments, the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs. In some embodiments, the ligation step is performed after the conversion step. In some embodiments, the ligation step is performed prior to or after end repair. In some embodiments, the ligation step is performed after end repair. In some embodiments, the ligation step is performed prior to or after A-tailing. In some embodiments, the ligation step is performed after A-tailing. In some embodiments, the ligation step is performed prior to or after end repair and prior to or after A-tailing. In some embodiments, the ligation step is performed after end repair and after A-tailing. In some embodiments, the ligation step is performed prior to or after digesting at least a portion of the DNA of the first portion of the converted DNA sample using a single-stranded DNA (ssDNA) nuclease. In some embodiments, the ligation step is performed after digesting at least a portion of the DNA of the first portion of the converted DNA sample using a single-stranded DNA (ssDNA) nuclease. In some embodiments, the ligation step is performed after denaturing DNA (e.g., DNA of the first portion, DNA of the third portion, or the DNA of the first portion and DNA of the third portion). In some embodiments, the ligation step is performed at about 1 degree Celsius to about 30 degrees Celsius, about 5 degrees Celsius to about 25 degrees Celsius, about 10 degrees Celsius to about 25 degrees Celsius, about 10 degrees Celsius to about 20 degrees Celsius, or about 20 degrees Celsius.

[0234] In some embodiments, adapters are ligated to end-repaired DNA molecules or the adapters are ligated to the DNA molecule or a plurality of DNA molecules. In some such embodiments, the ligation reaction also seals nicks present in the end-repaired DNA.Attorney Docket No. GH0197WO

[0235] In some embodiments, one or more adapters are preferentially ligated to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the differential likelihood that the one or more adapters ligate to the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% greater. For example, a differential likelihood of 50% would mean that 75% of ligation is to the second portion and 25% is to the first portion (75% - 25% = 50%). In some embodiments, one or more adapters are preferentially ligated to the DNA of the second and third portions of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the differential likelihood that the one or more adapters ligate to the second and third portions of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% greater. In some embodiments, one or more adapters are preferentially ligated to the DNA of the second portion of the converted DNA sample relative to the DNA of the first and third portions of the converted DNA sample. In some embodiments, the differential likelihood that the one or more adapters ligate to the second portion of the converted DNA sample relative to the DNA of the first and third portions of the converted DNA sample is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% greater.

[0236] DNA ligase and adapters are added to ligate DNA molecules in the sample with an adapter on one or both ends, i.e. to form adapted DNA. As used herein, “adapter” refers to short nucleic acids (e.g., less than about 500, less than about 100 or less than about 50 nucleotides in length, or be 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases from end to end) that are typically at least partially double-stranded and can be ligated to the end of a given sample DNA molecule. In some instances, two adapters can be ligated to a single sample DNA molecule, with one adapter ligated to each end of the sample nucleic acid molecule.

[0237] In some embodiments, the ligase used in ligation reactions can act on both single strand DNA nicks and double stranded DNA ends. In some cases, the ligase is T4 DNA ligase or T3 DNA ligase. Adapters can include nucleic acid primer binding sites to permit amplification of a sample DNA molecule flanked by adapters at both ends, and / or a sequencing primer bindingAttorney Docket No. GH0197WO site, including primer binding sites for sequencing applications, such as various next generation sequencing (NGS) applications. Adapters can include a sequence for hybridizing to a solid support, e.g., a flow cell sequence. Adapters can also include binding sites for capture probes, such as an oligonucleotide attached to a flow cell support or the like. Adapters can also include sample indexes and / or molecular barcodes. These are typically positioned relative to amplification primer and sequencing primer binding sites, such that the sample index and / or molecular barcode is included in amplicons and sequencing reads of a given DNA molecule. Adapters of the same or different sequence can be linked to the respective ends of a sample DNA molecule. In some cases, adapters of the same or different sequence are linked to the respective ends of the DNA molecule except that the sample index and / or molecular barcode differs in its sequence. In some embodiments, the adapter is a Y-shaped adapter in which one end is blunt ended or tailed as described herein, for joining to a nucleic acid molecule, which is also blunt ended or tailed with one or more complementary nucleotides to those in the tail of the adapter. In another exemplary embodiment, an adapter is a bell-shaped adapter that includes a blunt or tailed end for joining to a DNA molecule to be analyzed. Other exemplary adapters include T-tailed, C- tailed or hairpin shaped adapters. For example, a hairpin shaped adaptor can comprise a complementary double stranded portion and a loop portion, where the double stranded portion can be attached (e.g. ligated) to a double-stranded polynucleotide. Hairpin shaped sequencing adaptors can be attached to both ends of a polynucleotide fragment to generate a circular molecule, which can be sequenced multiple times. The adapters used in the methods of the present disclosure comprise one or more known modified nucleosides, such as methylated nucleosides. In some embodiments, the modified nucleosides comprise modification resistant cytosines. In some embodiments, each cytosine in each adapter is a modification resistant cytosine. In some embodiments, the modification resistant cytosine is a deamination resistant cytosine. In some embodiments, the deamination resistant cytosine comprises 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5-hydroxymethylcytosine (5hmC), glucosylated5- hydroxymethylcytosine (5ghmC), cytosine 5-methylenesulfonate (CMS), or N4-modified cytosine. In some embodiments, the adapters are resistant to digestion by a methylation resistant restriction enzyme (MSRE). In some embodiments, the MSRE digestion-resistant adapters comprise one or more methylated nucleotides, comprise one or more nucleotide analogs resistant to methylation sensitive restriction enzymes, or do not comprise a nucleotide sequence recognized by the MSRE. In some embodiments, the one or more methylated nucleotides in theAttorney Docket No. GH0197WO MSRE digestion-resistant adapters comprise 5-methylcytosine and / or 5-hydroxymethylcytosine. In some embodiments, the adapters are resistant to digestion by a methylation dependent restriction enzyme (MDRE). In some embodiments, the MDRE digestion-resistant adapters comprise one or more unmethylated nucleotides, comprise one or more nucleotide analogs resistant to methylation dependent restriction enzymes, or do not comprise a nucleotide sequence recognized by the MDRE.

[0238] In instances where two adapters are ligated to a sample nucleic acid (one at each end), either or both of the adapters may comprise one or more known modified nucleosides. Typically, the primer binding site(s), sequencing primer binding site(s), sample index(es) and / or molecular barcode(s), if present, do not comprise the known modified nucleosides that change base pairing specificity as a result of the conversion procedure.

[0239] In some embodiments, adapters may be added to the DNA or a subsample thereof. Adapters can be ligated to DNA at any point in the methods herein. In some embodiments, adapters are ligated to the DNA in a sample, to DNA after contacting the DNA with a methyl- sensitive deaminase, or to DNA in a converted sample. In some embodiments, adapters are ligated to the DNA in the sample after contacting the DNA with a methyl-sensitive deaminase. In some embodiments, adapters are ligated to the DNA in the sample after end repair and / or A- tailing reactions. In some embodiments, adapters are ligated to the DNA in a converted sample after end repair and / or A-tailing reactions. In some embodiments, adapters are ligated to the DNA in a sample or DNA in a converted sample after to digesting DNA using an ssDNA nuclease. In some embodiments, adapters are ligated to the DNA of a sample or subsample thereof prior to annealing primers to the DNA for capture probe generation. In some such embodiments, the adapter-ligated DNA is amplified prior to annealing primers to the DNA for capture probe generation. In some embodiments, adapters are ligated to the DNA of a sample or subsample thereof before the DNA is contacted with the capture probes. In some embodiments, the DNA to which the adapters are ligated is in the same sample or subsample as the DNA used as a template to generate capture probes. In some embodiments, the DNA to which the adapters are ligated is in a different sample or subsample, e.g., a second sample or a second subsample of a first sample, than the DNA used as a template to generate capture probes. In some embodiments, the adapters ligated to DNA captured by the capture probes.

[0240] In some embodiments, the primers used to generate capture probes are not complementary to adapters, and the resulting capture probes therefore do not comprise adapters.Attorney Docket No. GH0197WO Adapter-ligated DNA can therefore be selectively amplified in the presence of capture probes that do not comprise adapters. Similarly, adapter-ligated DNA can be separated from DNA that does not comprise adapters.

[0241] In some embodiments, the disclosed methods comprise analyzing DNA in a sample. In such methods, adapters may be added to the DNA. This may be done concurrently with an amplification procedure, e.g., by providing the adapters in a 5’ portion of a primer (where PCR is used, this can be referred to as library prep-PCR or LP-PCR), before, or after an amplification step. In some embodiments, adapters are added by other approaches, such as ligation. In some such methods, first adapters are added to the 3’ ends of the nucleic acids by ligation, which may include ligation to single-stranded DNA. In some embodiments, prior to any partitioning or capturing steps, first adapters are added to the nucleic acids by ligation, which may include ligation to single-stranded DNA (e.g., to the 3’ ends thereof). In some embodiments, the capture probes can be isolated after partitioning and ligation. For example, the hypomethylated partition can be ligated with adapters and a portion of the ligated hypomethylated partition can then be used to generate the capture probes for rearrangements. The adapter can be used as a priming site for second-strand synthesis, e.g., using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3’ end of the second strand of the now double-stranded molecule. In some embodiments, the first adapter comprises an affinity tag, such as biotin, and nucleic acid ligated to the first adapter is bound to a solid support (e.g., bead), which may comprise a binding partner for the affinity tag such as streptavidin. For further discussion of a related procedure, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercial kits for sequencing library preparation compatible with single- stranded nucleic acids are available, e.g., the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adapter ligation, nucleic acids are amplified.

[0242] In some embodiments, the single-stranded DNA library preparation is performed in a one-step combined phosphorylation / ligation reaction, e.g., as described in Troll et al., BMC Genomics, 20:1023 (2019), available at https: / / doi.org / 10.1186 / s12864-019-6355-0. This method, called Single Reaction Single-stranded LibrarY (“SRSLY,”) can be performed without end-polishing. SRSLY may be useful for converting short and fragmented DNA molecules, e.g., cfDNA fragments, into sequencing libraries while retaining native lengths and ends. The SRSLY method can create sequencing libraries (e.g., Illumina sequencing libraries) from fragmented or degraded template (input) DNA. In particular embodiments, template DNA is first heatAttorney Docket No. GH0197WO denatured and then immediately cold shocked to render the template DNA molecules single- stranded. The DNA can be maintained as single-stranded throughout the ligation reaction by the inclusion of a thermostable single-stranded binding protein (SSB). Next, the template DNA, which at this point can be single-stranded and coated with SSB, is placed in a phosphorylation / ligation dual reaction with directional dsDNA NGS adapters that contain single- stranded overhangs. Both the forward and reverse sequencing adapters can share similar structures but differ in which termini is unblocked in order to facilitate proper ligations. Both sequencing adapters can comprise a dsDNA portion and a single-stranded splint overhang of random nucleotides that occurs on the 3-prime terminus of the bottom strand of the forward adapter and the 5-prime terminus of the bottom strand of the reverse adapter. In this way, the forward adapter (e.g., (P5) Illumina adapter) can be delivered to the 5-prime end of template molecules and the reverse adapter (e.g., (P7) Illumina adapter) is delivered to the 3-prime end of template molecules. Thus, the native polarity of input DNA molecules can be retained.

[0243] During the dual phosphorylation / ligation reaction, T4 Polynucleotide Kinase (PNK) can be used to prepare template DNA termini for ligation by phosphorylating 5-prime termini and dephosphorylating 3-prime termini. T4 PNK works on both ssDNA and dsDNA molecules and has no activity on the phosphorylation state of proteins. Simultaneously, the random nucleotides of the splint adapter can be annealed to the single-stranded template molecule. This creates a short, localized dsDNA molecule, enabling ligation of template to adapter with a ligase such as T4 DNA ligase, which has high ligation efficiency on dsDNA templates but low efficiency on ssDNA. After the single phosphorylation / ligation reaction is complete, the library DNA can be, e.g., purified and placed directly into standard NGS indexing PCR, compatible with both traditional single or dual index primers.

[0244] In some embodiments, the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e.g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags. Adapters, whether bearing the same or different tags, can include the same or different primer binding sites, but preferably adapters include the same primer binding site.

[0245] In some embodiments, following attachment of adapters, the nucleic acids are subject to amplification. The amplification can use, e.g., universal primers that recognize primer binding sites in the adapters.Attorney Docket No. GH0197WO

[0246] In some embodiments, following attachment of adapters, the DNA or a subsample or portion of the DNA is partitioned, comprising contacting the DNA with an agent that preferentially binds to nucleic acids bearing an epigenetic modification. The nucleic acids are partitioned into at least two partitioned subsamples differing in the extent to which the nucleic acids bear the modification from binding to the agents. For example, if the agent has affinity for nucleic acids bearing the modification, nucleic acids overrepresented in the modification (compared with median representation in the population) preferentially bind to the agent, whereas nucleic acids underrepresented for the modification do not bind or are more easily eluted from the agent. The nucleic acids can then be amplified from primers binding to the primer binding sites within the adapters. Partitioning may be performed instead before adapter attachment, in which case the adapters may comprise differential tags that include a component that identifies which partition a molecule occurred in.

[0247] In some embodiments, the nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags. The molecules are amplified. E. Molecular Tagging

[0248] In some embodiments, the DNA molecules of the sample may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”). In some embodiments, the DNA molecules of the sample comprise barcodes.

[0249] Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated. For example, DNA molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), a partition tag (which distinguishes molecules in one partition from those in a different partition) and / or a molecular tag / molecular barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios)).

[0250] Tagging strategies can be divided into unique tagging and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample bear a different tag, so that reads can be assigned to original molecules based on tag information alone. Tags used in such methods are sometimes referred to as “unique tags”. In non-unique tagging, different molecules in the same sample can bear the same tag, so that other information in addition to tag information is used to assign a sequence read to an original molecule. Such information may include start and stop coordinate, coordinate to which the molecule maps, startAttorney Docket No. GH0197WO or stop coordinate alone, etc. Tags used in such methods are sometimes referred to as “non- unique tags”. Accordingly, it is not necessary to uniquely tag every molecule in a sample. It suffices to uniquely tag molecules falling within an identifiable class within a sample. Thus, molecules in different identifiable families can bear the same tag without loss of information about the identity of the tagged molecule.

[0251] In certain embodiments, a tag can comprise one or a combination of barcodes. As used herein, the term “barcode” refers to a nucleic acid molecule having a particular nucleotide sequence, or to the nucleotide sequence, itself, depending on context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain Hamming distance, as desired for the specific purpose. So, for example, a molecular barcode can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule. Additionally or alternatively, for different partitions and / or samples, different sets of molecular barcodes, molecular tags, or molecular indexes can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition and / or sample to which they correspond based the set of which they are a member. For example, barcodes can be used to allow the origin of the DNA (e.g., the subject, biological sample (e.g., samples collected at various time points), enriched DNA sample (e.g., enriched DNA comprising an epigenetic target region set or enriched DNA comprising a sequence-variable target region set), partition, or similar) to be identified, e.g., following pooling of a plurality of samples for parallel sequencing. Tags comprising barcodes can be incorporated into or otherwise joined to adapters. Tags can be incorporated by ligation, overlap extension PCR among other methods. Tags can be used to label the individual polynucleotide population partitions so as to correlate the tag (or tags) with a specific partition. Alternatively, tags can be used in embodiments of the disclosure that do not employ a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated for the set of tags used to label other partitions. In some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’l Acad Sci USA 108: 9530-9535 (2011), Kou et al.,Attorney Docket No. GH0197WO PLoS ONE,11: e0146638 (2016)) or used as non-unique molecule identifiers, for example as described in US Pat. No.9,598,731. Similarly, in some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as non- unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).

[0252] Tags may be incorporated into or otherwise joined to adapters by chemical synthesis, ligation (e.g., as described above, e.g. by blunt-end ligation or sticky-end ligation), or overlap extension polymerase chain reaction (PCR), among other methods. Such adapters are ultimately joined to the sample DNA molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce sample indexes to a nucleic acid molecule using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after any conversion procedure. In the case of molecular barcodes and / or sample indexes being introduced through amplification processes, the conversion step will occur before the molecular barcodes and / or sample indexes are introduced. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after sequence capturing steps, if present, are performed. In some embodiments, only the molecular barcodes are introduced prior to probe capturing and the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, both the molecular barcodes and the sample indexes are introduced prior to performing probe-based capturing steps, if present. In some embodiments, the sample indexes are introduced after sequence capturing steps are performed, if present. In some embodiments, sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).

[0253] In some embodiments, the tags may be located at one end or at both ends of the sample DNA molecule. In some embodiments, tags are predetermined or random or semi- random sequence oligonucleotides. In some embodiments, the tag(s) may together be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length. Typically tags are about 5 to 20 or 6 to 15 nucleotides in length. The tags may be linked to sample DNA molecules randomly or non-randomly.Attorney Docket No. GH0197WO

[0254] In some embodiments, each sample or partition (discussed below) is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or sub-sample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation as part of an adapter) to individual molecules such that the combination of the molecular barcode and the sequence it may be attached to creates a unique sequence that may be individually tracked. Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original DNA molecule in the sample, start and stop genomic positions corresponding to the sequence of the original DNA molecule in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original DNA molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand, and / or a complementary strand.

[0255] In certain embodiments of non-unique tagging, the number of different tags used can be sufficient that there is a very high likelihood (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999% that all DNA molecules of a particular group bear a different tag. It is to be noted that when barcodes are used as tags, and when barcodes are attached, e.g., randomly, to both ends of a molecule, the combination of barcodes, together, can constitute a tag. This number, in term, is a function of the number of molecules falling into the calls. ForAttorney Docket No. GH0197WO example, the class may be all molecules mapping to the same start-stop position on a reference genome. The class may be all molecules mapping across a particular genetic locus, e.g., a particular base or a particular region (e.g., up to 100 bases or a gene or an exon of a gene).

[0256] In certain embodiments, the number of different tags used to uniquely identify a number of molecules, z, in a class can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, 9*z, 10*z, 11 *z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z or 100*z (e.g., lower limit) and any of 100,000*z, 10,000*z, 1000*z or 100*z (e.g., upper limit). In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule. Alternatively, from about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20-50 x 20- 50 molecular barcode sequences (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the target molecule) can be used. Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of molecules have the same combinations of molecular barcodes. For example, in a sample of about 5 ng to 30 ng of cell free DNA, one expects around 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules having any start coordinate to share the same stop coordinate. Accordingly, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all such molecules. To uniquely tag all 3000 molecules mapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0257] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos.20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only endogenous sequence information (e.g., start and / or stopAttorney Docket No. GH0197WO positions, sub-sequences of one or both ends of a sequence, and / or lengths). Tags can be linked to sample nucleic acids randomly or non-randomly.

[0258] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, the unique tags may be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the unique tags may be loaded so that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the average number of unique tags loaded per sample genome is less than, or greater than, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags per genome sample.

[0259] In some embodiments a format uses 20-50 different tags (e.g., barcodes) ligated to both ends of target nucleic acids. For example, 35 different tags (e.g., barcodes) ligated to both ends of target molecules creating 35 x 35 permutations, which equals 1225 for 35 tags. Such numbers of tags are sufficient so that different molecules having the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different combinations of tags. Other barcode combinations include any number between 10 and 500, e.g., about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0260] In some cases, unique tags may be predetermined or random or semi-random sequence oligonucleotides. In other cases, a plurality of barcodes may be used such that barcodes are not necessarily unique to one another in the plurality. In this example, barcodes may be ligated to individual molecules such that the combination of the barcode and the sequence it may be ligated to creates a unique sequence that may be individually tracked. As described herein, detection of non-unique barcodes in combination with sequence data of beginning (start) and end (stop) portions of sequence reads may allow assignment of a unique identity to a particular molecule. The length or number of base pairs, of an individual sequence read may also be used to assign a unique identity to such a molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand.Attorney Docket No. GH0197WO

[0261] In some embodiments, the method includes adding one or more internal control DNAs and forward and reverse primers for amplifying the internal control DNAs. The internal control DNAs may be added before amplification using the primers that anneal upstream and downstream of the rearrangement breakpoints. The forward and reverse primers for amplifying the internal control DNAs may be included with, or added at the same time as, the primers that anneal upstream and downstream of the rearrangement breakpoints. The internal control DNAs may comprise or consist of sequences that do not occur in the genome of the subject, or that do not occur in the genome of the species of which the subject is a member (e.g., the human genome). The forward and / or reverse primers for amplifying the internal control DNAs may comprise sequences that are not complementary to any sequence in the genome of the subject, e.g., the human genome. The internal control DNAs may be used to ensure that the amplification process proceeded as designed. As such, the method may comprise detecting (e.g., sequencing) molecules amplified from and / or captured by the one or more internal control DNAs. The method can comprise comparing an amount of internal control DNAs (e.g., number of molecules or reads detected that correspond to an internal control DNA sequence) to a predetermined threshold, and either rejecting sequencing results if the predetermined threshold is not met or accepting sequencing results if the predetermined threshold is met. The predetermined threshold may be established, e.g., based on historical data or by testing the method on samples of DNA from test subjects, such as healthy volunteers. For example, amplification and detection of the one or more internal control DNAs provides confirmation that the amplification process proceeded properly, thus reducing the likelihood of a false negative. F. Conversion; Contacting the DNA with a Deaminase

[0262] The methods disclosed herein can comprise contacting DNA in a sample with a methyl-sensitive deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. In some embodiments, DNA of a third portion of the converted DNA samples comprises both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA. The methods disclosed herein can further comprise contacting end repaired DNA, optionally further comprising an A-tailing reaction, with a methyl-sensitiveAttorney Docket No. GH0197WO deaminase, thereby providing a converted sample in which unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, the methyl-sensitive deaminase is a dsDNA deaminase and / or has ssDNA deaminase activity. In some embodiments, the methyl-sensitive deaminase is a CpG-specific deaminase. This step of contacting the DNA in the sample with a methyl-sensitive deaminase can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. For an exemplary description of conversion using a methyl-sensitive deaminase, see, e.g., Vaisvila et al. (2023) Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra- low input DNA. bioRxiv; DOI: 10.1101 / 2023.06.29.547047, available at https: / / www.biorxiv.org / content / 10.1101 / 2023.06.29.547047v1. One or more adapters are then preferentially ligated to the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample comprising converted UpGs and is denatured to form single-stranded DNA. The DNA with ligated adapters is then amplified, partitioned, eluted, and / or enriched. In some embodiments, the DNA with ligated adapters is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of end-repair prior to the conversion step or after the conversion step.

[0263] In some embodiments, the denaturing of the DNA (e.g., DNA of the first portion, DNA of the third portion, or the DNA of the first portion and DNA of the third portion) comprises heat denaturing. In some embodiments, heat denaturing comprises heating DNA at a temperature of about 70 degrees Celsius to about 100 degrees Celsius, about 80 degrees Celsius to about 100 degrees Celsius, about 90 degrees Celsius to about 100 degrees Celsius, or about 88 degrees Celsius, or about 95 degrees Celsius for about 1 minute to about 15 minutes, about 2.5 minutes to about 10 minutes, or about 5 minutes. For an exemplary description of thermal denaturation of DNA to selectively render GC-poor DNA single stranded while keeping GC-rich DNA double stranded, see, e.g., Cheruba et al., Sci Adv.8, eabn4030 (2022). In some embodiments, the denaturing of the DNA (e.g., DNA of the first portion, DNA of the third portion, or the DNA of the first portion and DNA of the third portion) comprises contacting the DNA with a DNA helicase, including e.g., a UvrD helicase.

[0264] Deamination of unmethylated cytosines converts cytosines to uracils, which reduces the GC content of DNA. In some embodiments, regions of double-stranded DNA (e.g.,Attorney Docket No. GH0197WO double-stranded DNA of the first portion, double-stranded DNA of the third portion, or double- stranded DNA of the first portion and double-stranded DNA of the third portion) with a lower degree of methylation relative to double-stranded DNA with regions with a relatively higher degree of methylation, is denatured following deamination of unmethylated cytosines. The denaturation can be complete denaturation or partial denaturation. In some embodiments, regions of double-stranded DNA with a relatively higher degree of methylation, relative to regions of double-stranded DNA with a relatively lower degree of methylation, are not denatured or are denatured to a lesser extent. In some embodiments, regions of double-stranded DNA with a lower amount of CpG content, relative to double-stranded DNA with regions of relatively high CpG content, are denatured to a greater extent, in which the denaturation is complete denaturation or partial denaturation. In some embodiments, regions of double-stranded DNA with a relatively high amount of CpG content, relative to double-stranded DNA with regions of relatively low CpG content, are not denatured or are partially denatured.

[0265] In some embodiments, the DNA (e.g., DNA of the first portion, DNA of the third portion, or the DNA of the first portion and DNA of the third portion) can be contacted with a single-stranded DNA binding protein. In some embodiments, the contacting the DNA with a single-stranded DNA binding protein (SSB) occurs after denaturing (e.g., heating) the DNA. In some embodiments, the contacting the DNA with a single-stranded DNA binding protein (SSB) occurs concurrently with denaturing (e.g., heating) the DNA; for example, a thermostable SSB can be used. In some embodiments, the contacting the DNA with a single-stranded DNA binding protein (SSB) occurs prior to denaturing (e.g., heating) the DNA. In some embodiments, the single-stranded DNA binding protein is E. coli single single-stranded binding protein. In some embodiments, the single-stranded DNA binding protein is human single-stranded DNA binding protein. In some embodiments, the single-stranded DNA binding protein is a thermostable single-stranded binding protein. In some embodiments, the binding of the SSB to denatured (i.e., single-stranded DNA), can inhibit the reforming of double-stranded DNA.

[0266] In some embodiments, the contacting the DNA in the sample with a methyl- sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, all unmethylated CpGs in the sample can be converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of unmethylated CpGs are not converted to UpGs using the methyl-sensitiveAttorney Docket No. GH0197WO deaminase. In some embodiments, at least a portion of methylated CpGs can be converted to UpGs using the methyl-sensitive deaminase.

[0267] As outlined below, there are various methods of detecting and / or identifying modified nucleosides that rely on a conversion procedure that changes the base-pairing specificity of a nucleoside, based on the modification status of the nucleosides. These changes of base-pairing specificity can then be detected, and thus the modification status of the nucleoside inferred, by sequencing.

[0268] In some embodiments, the conversion procedure used in the methods of the disclosure is one that changes the base pairing specificity of an unmodified nucleoside (e.g. cytosine), but does not change the base pairing specificity of the corresponding modified nucleoside (e.g. methylated cytosine, such as 5hmC and / or 5mC) or does not change the base pairing specificity of any modified nucleoside (e.g. modified cytosine, adenosine, guanosine and thymidine (or uracil)). In methods that require denaturation for conversion, failure to denature a DNA molecule will result in non-conversion of all bases in the DNA molecule. As biological changes in methylation are predominantly concerted to a localized regions of interest, these non- random (localized) non-conversion events can appear as false negatives (non-methylated regions). Random non-conversion methods can maximally affect a low percent of bases within a region, and thus the specificity of methylation change detection can be maximized (reduce false positives) by placing a threshold on percentage of bases within a region that are methylated / non- methylated. Hence, in some cases, a conversion procedure that does not involve denaturation is preferred.

[0269] The skilled person can select a suitable method according to their needs, including which nucleoside modifications are to be detected and / or identified and which type of modified base is used in the end repair reaction.

[0270] The methods described herein could in principle use any suitable methyl-sensitive enzymatic conversion procedure that changes the base-pairing specificity of the unmodified cytosine and thereby allows the modified base to be distinguished from the corresponding modified cytosine and / or other types of modification when sequenced. For example, any enzymatic conversion procedure could be used allowing 5-methylcytosine (m5c or 5-mC or 5mC) to be distinguished from unmodified cytosine. In particular embodiments, the conversion procedure converts unmodified (e.g., unmethylated) cytosines to uracils using a methyl-sensitive deaminase.Attorney Docket No. GH0197WO

[0271] In some embodiments, the conversion procedure comprises enzymatic conversion of a nucleobase using a non-specific, modification-sensitive double-stranded DNA deaminase, e.g., as in SEM-seq. See, e.g., Vaisvila et al. (2023) Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA. Mol Cell. 2024.84(5):854-866.e7. SEM-Seq employs a non-specific, modification-sensitive double- stranded DNA deaminase (e.g., modification-sensitive DNA deaminase A (MsddA)) in a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method that deaminates unmodified cytosines. Accordingly, SEM-seq does not require the TET2 and T4-βGT or 5- hydroxymethylcytosine carbamoyltransferase protection and denaturing steps that are of use, e.g., in APOBEC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5-caC). In SEM-seq, unmodified cytosines in the DNA are deaminated to uracil and is read as “T” during sequencing. Modified cytosines (e.g., 5mC) are not converted and are read as “C” during sequencing. Cytosines that are read as thymines are identified as unmodified (e.g., unmethylated) cytosines or as thymines in the DNA. Performing SEM-seq conversion thus facilitates identifying positions containing 5mC using the sequence reads obtained. In some embodiments, the conversion procedure comprises enzymatic conversion of unmodified cytosine using MsddA or a modification-sensitive DNA deaminase A (MsddA)-like deaminase. For an exemplary description of MsddA and MsddA-like deaminases, see, e.g., Vaisvila et al. Mol Cell.2024 Mar 7;84(5):854-866.e7, which illustrates in Fig.2A-C that MsddA-like deaminases have reduced activity on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine in dsDNA, e.g., a reduction of about 75%, 80%, or more on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine (e.g., using assay conditions as described in Vaisvila et al., such as analysis of deamination of C in E. coli or lambda dcm- DNA, deamination of 5mC in XP12 phage DNA, deamination of 5hmC in a C-hydroxymethylated adenovirus PCR fragment or fully C-hydroxymethylated T4147 phage DNA, and deamination of 5gmC in alpha-glucosyltransferase knockout (AGT-) T4 phage DNA. Deamination can be performed by contacting substrate DNA with deaminase and analyzed using NGS as follows: 50 ng of unmodified E. coli C2566 genomic DNA can be combined with the control DNAs (about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment), sheared to about 300 bp and ligated to pyrrolo-dC adapters with 1 uL of in vitro synthesized deaminase (e.g., synthesized using the PURExpress In Vitro Protein Synthesis kit (NEB, Ipswich, MA)Attorney Docket No. GH0197WO following manufacturer’s recommendations with 100-400 ng of PCR fragment template DNA containing codon-optimized deaminase coding sequence and T7 promoter and terminator). Exemplary deamination reaction conditions are 50 mM Bis-Tris pH 6.0, 0.1% Triton X-100 for 1 hour at 37 degrees C. After the deamination reaction, 1 uL of Thermolabile Proteinase K (NEB, Ipswich, MA) can be added and incubated for 30 min at 37 degrees C and then the Proteinase K can be heat inactivated at 60 degrees C for 10 minutes. The deaminated product can then be used for library amplification using the NEBNext Q5U Master Mix (New England Biolabs, Ipswich, MA, USA) with 5mMof NEBNext Unique Dual Index Primers. The resulting library can be purified using 1X NEBNext Sample Purification Beads according to the manufacturer’s instructions and the purified library can be analyzed and quantified by an Agilent Bioanalyzer 2100 DNA Highsensitivity chip. The libraries can be sequenced using the Illumina NextSeq and NovaSeq platforms. Paired-end sequencing of 75 cycles (2 x 75 bp) can be performed for all the sequencing runs. Base calling and demultiplexing can be carried out with the standard Illumina pipeline.

[0272] In some embodiments, the methyl-sensitive deaminase comprises MsddA. In some embodiments, the methyl-sensitive deaminase comprises any one or more of MsddA, AshDa01, MGYPDa21, PpDa03, SbDa01, BlDa01, PpDa04, CsDa01, MGYPDa22, FlDa01, MGYPDa24, AaDa02, MmgDa01, PbDa01, BcDa02, LsfDa01, SmgDa01, XcDa01, KsDa01, PwDa01, CaDa01, SrDa01, NgDa01, NsDa01, SzDa01, SpDa01, AdDa01, MGYPDa23, WWTPDa07, PdDa01, MGYPDa25, MGYPDa26, DaDa01, EcDa01, EcDa02, NgDa02, PaDa01, AsDa01, HgmDa01, MsDa02, XinDa01, XjaDa01, RhDa01, MGYPDa04, MGYPDa05, BaDa01, WWTPDa04, PbDa02, CrDa01, MGYPDa15, MGYPDa16, MGYPDa17, BaDa02, VsDa01, MGYPDa18, MGYPDa19, HmDa06, MmgDa02, HgmDa02, CgmDa01, FbiDa01, PvmDa01, SjDa01, HmDa02, DddA, RaDa01, MGYPDa624, AvDa01, CbDa01, PfDa01, NoDa01, or SsdA. In some embodiments, the amino acid sequence of the methyl- sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, which is the amino acid sequence of MsddA (Accession: MGYP001011792517; database: MGnify): QDPIGLLGGNNLYQFAPNAQGWVDWLGLSYGFNIKANRWTNFETGKFVQKPSTLSGQA QAVHNLAGGLKPSKISIEKSTVAIFEVQLRDGSKTLYAAGSSGVLNPRQRALLKQIGVPE KNILFGKGSVLPVTKVNSNINHAEQIILRNIPKDAKVTRKGISWSSKHKNAPCSKCQSSV DGAGGVYD (SEQ ID NO: 1).Attorney Docket No. GH0197WO

[0273] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7, which is the amino acid sequence of XcDa01 (Accession: B0RYS5; database: UniProt): MLKRIMRRAALLLALAWPAAGLAETVEYIHTDALGSPVAITDAAGNVIERTVYEPYGG MVNKAAVDGPGFTGHVADSATGLSYMQQRYYDPQVGRLLSVDPVMAHGNPLGAFNR YWYANDNPYRFTDPDGRQTIPSENKLGSSDPAVINDFHQAQGEHAAITLNVALDLFLSQ FGDGPGGAAAGVGGVVAKSAEAVSALTRAREIQGALKPITQSKVTTAVTETAEGVRVV TSSEGRLRPAQRAALQVGEVAGKGAARTHAEVNGVNAARDMGLTPTSVSPSRTACPGC QEAMKKLEIPIKDK (SEQ ID NO: 7).

[0274] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8, which is the amino acid sequence of KsDa01 (Accession: E4NEH0; database: UniProt): MAVTGSLVGAPPAFAEKPPKVWVPPTTPLQEVKSVSGRSDNVTGTAAVGGVPREWKR AAVAPAKAGSATVDVTDVSAEQEAARRLGGGAAPAAGGKAKVKAGDLPVFLTDLGA ANDRDRAAGVPSVKVDLLDEGRAKAAGSTGPVVRLEGAGGAAKGGHKLQVSLDVKA LTGNAMWSDRARLVLLPECALTTPDAEQCRTSTPVPSTVDPLSGLLTADVQLPEAPAVP AGTATQAGFVQQAGFVQQASAASSGAMVLAAQAAPSGSSGSFAATAIAPSAAWSAGSG SGNFTYSYTFDMPTAVGGAAPSVVLGYDSSSVDGRTASTNSQSGWVGDGWDYSAGSIS RSYKACLKSGIDMSGDECWAGQALQLNLAGHSGTVVKDDTTGVLRLQGDDGTLVTPL TGLANGAWNGEGFKVTTTDGTQYYFGANHLPGGDNTDPASNSVNTVPVYHPHAGDPC YSSSTGLASWCQMGWQWNLDYIVDLHGNLIKYQYAKESNSYARGGGQNNGTGTRTA YDRYSNVSSIQYGLRLSDQIASKGAGNPAAKIDFTTAERCFASGSITCSDAQRVVANQSS WPDTPLDQACTASGTCTNYGPSFWTSKRLTGIQTTVYVGGAARVVDSWALNQVFSDPG DGNAPTLWLSSIQRTGKNGQADIALPAVSFTAMQMANRVDGLVPAAPQFNRPRMKEIT TETGGRITVNYKPAECSRVNNTMPASEAGNTMACMPVKWVPPGSQAGTKPIDDWFHK YLVSSIVESSLVTSSIVAKETQYAYGGGAAWHRNDSEFADDDTRTYDNFRGYQSVTTTT GSGSDGPRTKSVSTFLRGMGGQVTDSWGSTITDAEELAGFVRETQNYESDAAGAKVIG GELTTPWLSKITATHTQSGNLPKVTARYINTANVKNRSLLSNGQWRTTERSATYDEDHA GRLVTMDVLGDTTLSGLPQRQCTAVTYASGSNPALVEFPSRMLTLAGACAQTPSATNTI SDALTYYDGQALGVIGDKGDQTETRVLESYGTGNTPNYKVTAKAQFDAYGRQTSTTDPAttorney Docket No. GH0197WO NRTDTAHPNGATTSTAYTPATGELPSKVEVTNPLGWKSTTLVDPARGLPTKATDENGRT TLETYDALGRLTAVWLPGRDPNDLDGNDTPNRKFTYALNGSTSPSVVTSESIREDRTYA KSLQIYDGLGRVRQSQSIPVQGQVGRLISESFYDSHGWQIKSTPSYYNADSGPIATVYLPE DSEVPTQTWNEYDGLGRITASKFISYGQEQWRSTASYPGTERTDATPPPGGYATTTLTD ATGAAVEVRQYKAGTPTGPYDATTYGYDVAGKPTWVQDSSTPAHRWTYTYDLLGQRT GSTDPDTGTSATVYDKAGHAVTTTDARGKSTSITYDLLGRQVASYDGTSTTDASKKLIE WTYDSLALGKPAATTTYADVPNGTGKVAYVGAVTGYDIGYRPTGMSMTIPSVTGEEAL AKTYTWSTTYTPVLGLVKNTKLPAVGGLAAETVGNSYDVDGNFQTSSGLDYYVQDMQ YDAFGRPTRTTVGKAGKQVVSTQQYDPGTGNVVQSTLDKQTATTTHVDFTNYTYNKV GSMTSAKTIQDGTSTDLQCFTYDYLGRLSSAWTDKGTTTTAPAPSVQGIGNCTNATEPT ALTASARIGGPAPYWQTYSYDLTGNRTKLVQHDTTGAVVNDKQTDQTFAPGPNTATTD PTTGGGTGGPHALLTSKTSSNGTSKVTTSTYDATGNTTSLTSTSGTKTLTWNNQGKLAK VHDTAAPANDTSYVYDADGNQLVRRVGGKTTINLGTDELTLDTATGKTTATRYYAAP GGLTITRTADAAGATTLYYQASDPHGTAGVQMDAANLGVTRRPTDPFGNARGTQPGN GAWAGDKGFVGGTIEGTGFTNLGARQYDPATARFLSVDPLFDNSDPQSWNGYAYANN NPVDGSDPNGTCRVLDDGYCHGAPFIPVNHNSGDDGSGTDTSTSTSATGKAAHDASQE ALRQKQAADEALAKAKQAREELISKIVDVVGDLIGFNDARDCFTKGDVMGCINTALNF VPWAKVFKAVKIGIKAFKLWREGEKAYTAIRSAERVAKEAQQAFSVARKTEKEVAEAE QQAAKAAEADAAKTAKEADAAKADSAGTSESSASCPAGNSFPAGTLVQLADGTTKPID QLEPGDTVLATDPQTGETRAEPVTATITGHNDTEFTQLTLQGTESGDSLTVTSTAHHPY WDETSHQWVDALDVKAGHQLRTADGTLITVTALRTYSTQPSSAHNLTVANLHTYYVL AGATPVLVHNCGGELLDRARELYGTRADEASTVAVARVRSTSNPDKVETWVATERTGL PDEWKGGNAPLRGERYISGQGHAEATIMNRLGSDWEIVGMASSTRMCPTCFAQATGPG VGLTPSPIGKGAGVSRTGNTPWRVVLGGGG (SEQ ID NO: 8).

[0275] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9, which is the amino acid sequence of PwDa01 (Accession: UPI0001B0E918; database: UniParc): MREEILSRIARVGAMHAGNRPKPPPDLPEPGQGKPPTSPGKAIKHSSFWGALAGAAVGA LIAAAAFTVAAAAVAGAVALTGITGGAGLALVVGVVKLAVGFGAVFALGDLIGGVTNR VSAMVDSASPSSGAVKDGSKTVFVEGNPVSRAEIDAVLCDKHSGPQLIAQGSATVFVEG YYAARVGDKTVCGATIKEGASTVFFGSGQESPLSVADEFSGWEKALILAVEFLMPPSRGAttorney Docket No. GH0197WO LFRGLGKLFTKGPMAVLRGMKAGAAHALEGLRSAVQCAKNGFKESKGLARVTESIRGF LKDPVYIASGEVIEKRLDLELGQTVPLLFERTYRSGSPHSGLLGHGWHDSWSEVATVGR TEQGDIQVTLTLAQGYDIDFTFVVGATVVYCAEYPEFKLVKRHNGFHLWHRDSQTWRA FTAKQGDQLLLSAITDNHRNRIDFLRDPKGYLRKIRHSDGIELLLVWQGEFLRQVQRIDG GQKTVLAEYRQDEQGRLVEADAAHAYHLFYDYDSHHRLIRWHDNDQTWARYEYDPQ GRCVYTTCADGYLTANFEYLADRVVMIDGLGQRHEYGFNDLYLMAWEKSPLGHITHY EYDDVGNLLREISPAGRVVEFAYLDNSGLVSTFTDGSGHQWHYDYDHYERLIGITDPLG RSWAWQYDSKGNPLSLTGPDASEVRFAWNRYGLLTEVSDEQGRTQASLFYDHRHRLM SATDAESRTRQLRYDQQDRLTSWTRPDGATYRLGYRRASWKLPEQLLRPDDKQEQRQ YDKHNNLLNYTDGNGAVWAQTYGPFDLLTSRTDAEGRTWQYEYDKESQQLVCVTAP DGNRWQWWLDADARVIRERDMAGTETHYGYDEDGHCIAVRNGEGETRHFLYDGRGL LIKETAPDDTLHYRYDAAGRLIEVTSSTSHIQLEYDERDRVVQERNSGTAIRRHYQDTSH TVTRSLHWEGEEDSAALTSTFRYSATGELRQVQLPDGAELTLAHDSAGRESSRQSQAGF MQHREYDAMGWLTREMSGQAVDGRLQPTQTREYLYDGAGNLTGTRRNREAAGYQLD ASGRVLSVLSGGAGRSVNTEEQYRYTRSGLPQDATRLTEWQAGRLTQQDNTHYQYDK AGRLIRKQVVQPGYRPQVWHYRWDSRNQLRVVDTPAGERWFYRYDPFGRRIGKRCDQ THEDIRYLWDGDQIAEVRHYRDDELVSRRHWIHNGWELLVQQRQNADGRWETDFMTS SHNGEPQAAFNLAGELRWQAPRTNLWGQRYTDNTENLDPGLAFAGQYRDAESSLCYN RFRYYDPNGGYYISPDPVSIFGGDVNYGYAHNPLGWVDPFGLASCEHLNRMAKEVHDL AGGLKTPDTRAIRNSTVSIVEATVDGKKVLYAAGSGGRLNPRQVETLERLGVSRENIFK GARVTNEFSKLENHAERIILRNLPEGAVVNRWGISWGGLQRGIPCDHCAPFVRDAGGFF (SEQ ID NO: 9).

[0276] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, which is the amino acid sequence of CaDa01 (Accession: C7QJN3; database: UniProt): MATFAIGGRLRRSVAAVATAALGLAAFVAPTTAQAQAAAGPGSAAQHPASALGRSGFA MADPAPPPALPAPLDSSGPAQVQALEQQYQTDAQNDQQKYDQLAAEKGTLEQRAGDV QNRESSLETQATNLEDQSNALNSQAETLNSEIDAHNAEPHTFELPDEEAEYAAYNEEKA NLDEQKANLQNQIDALTAQSKKLQSDQAQADADQTQLETDVQTHNDAVSALEGDVGK LEAERQQILTQIDSLLQDYAGAEPGGEGAPLAAEGGDESEPAAAAAPSPGARALPSGGG DQSAPPRSTYAPVQNAPSGSGSGQAQAQAAPAPAPTQTPVTVTLAPSTVSGLPASEAENAttorney Docket No. GH0197WO LQPSETFDGLIPEANGDYAAEEIQPPAGESVPPAQKAFDNVVNKGGKASTRIGGRPATID KIVPEAAAPAENQGGDTPRPAKAAAPPARSSWVPAVNQPNPANGPPVSIDALKSLLDQQ GLGSDADQFDLEYSPTVLGQDGEPAYAVAPTDAAGNPELGAEGKPILRFSNLGLQNPEV AQDAFENEGLDVEPAQDDSPCPHSFSGATRVLMADGSTKAIAEVGVGDLVENAEPGGR AEVHRVDQVHTTTTDAAFVDVVVASAAPGGGGTLTGTANHPYYDATAGGFVDAGALR AGDRLQSAGGGQATVSGVHARFGPLVTYDLTIDGLHTYFVVAGSAPVLVHNCDGDLLD IAKDSATRASGYQKLAGSDWEVKNKTTSVIRARFPSGDPKNPWVYKNVVSSSGSGLSPA QISAIEANGDVAVTDNLEGFTHAEYNGLRYIDSMGGQPIAGGASRSVCTTICGPFIRGTD GNISGPVYQLEHGTKIRTFYWPGSTPG (SEQ ID NO: 10).

[0277] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11, which is the amino acid sequence of SrDa01 (Accession: D6Z8J1; database: UniProt): MSRPSQLRSTIDSATSSAKGQRPAEDAGVAGVRAATRDPFAEASSLLVHLVINRVPPIKH VLDKVTLDEGAAMALAQQWSDEFPAALREQRDALVSTRESISQNWEGEGASKTYQDR QRELEDLADSLAAACEGAGFLTSAVNELMVGVREQIIEWIAQLVSMLMRRLISVVATFW IPIVGEINEAAFIFEGVARVVETIERITSLITRVEGILGEVSSVAQVLGGSTQQVQQLGSAL GEVPQGLGASPLRALGRVGSVRQSGLTGSHGAVIRSVVPRRSKSPGGSAHAQSAGTSTR GLGSQTGATPSASSGTHPQGGAVNASTRSLSPDSGTRGPVTRSQTAAERAQSSNANAQA EQARLDARARDLHSRLVNARTGEPDTRAQNSRTSVAMSTREGRDVLAGGGRDLNPSQR QAAGPNDVVATQPGAHAEVTAINGARAAGLTPSQIAVSRPICDNCQDAIQQSGGTVHPD RMGATWP (SEQ ID NO: 11).

[0278] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, which is the amino acid sequence of NgDa01 (Accession: G3Z1X9; database: UniProt): MPNKEENARNGLPETDWRDAQYDMLYLPVTETVRYHYDFNGNRTATVLPDGRQINYL YYGSGHLHQISLDDEVITDIERDKLHREIYRTQGKLASRYELDPLGRLKRQIATLNDLTET GKGKTKVAAGYGQTAVKRSYGYDRTGNLTHSTDQRTGTTKFEYDKLGRITQAGNELF AFDPAHNILSDDLNTITDNRLKTYNGTTYYYDELGNLIHRELADGEVQNYFYDLHDQLV KAEIFKKDGTKETWSYTYDALGRRIGKGRLKTSQKVSETSFPHDLSGNDLENQTRFVWD GSHLLQEIHPDGRYTYIYADQNSYEPLAQVRDWTTVEDENRQQTRYFHCDQIGIPREMTAttorney Docket No. GH0197WO DKDGNLLWFGNYTGWGRLKEETKVTDSAYQPFRLQNQYADRETGLHYNFFRYYEPDA GRFVNQDPIGLWGGDNLYRFSSNIQIWIDPLGLACIPNPNRKNDEDLARRIDKLSDNLTE KNRDGFTTLALARVTLADGTSQIWIAQAGSSLSSKPSRRQQSLAGADEIIQNLHPAGRSR KDGNHLNDAERQLIREAKKRGAKIKSLGATKPMCGRCEKGARRAGILRRIITPIKSRHC (SEQ ID NO: 12).

[0279] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13, which is the amino acid sequence of NsDa01 (Accession: D3A5B8; database: UniProt): MAGGAVNRRYAYDKAGNLIQSADQRSGVLNYVYDKIGRIQEARNSQSGRSETFAFDPA HNILSDKAAEGKGNNLVSGNRLKEYNGIEYTYDALGNLIYRQLPNGENQYYQYDLENQ LVRAEIKKPAGNTEIWTYAYDPFGRRLSKERQDKLAWTSTDPKRTHFVWDGTRLLQEY TYKGSYTYIYTDQDSYEPLAQIFDNAKDSKQYLAYFHNDQISIPREMTDIHGNLLWYGE YTAWGRLKKDERVYKNAHQPFRLQNQYFDEETGLHYNLMRYYEPEAGRFVNQDPIGL LGGINLYQFSPNVQTWIDPLGLNAKLSEMAQSVHNLAGGLKTPDARAIEKSTVAIFEVEL KNGSKTLYAAGSSGRLNPRQRSLLEKLGVPKENILSGKKYTLPKDVVKNINHAEQIILRN IPDDAKVTRKGISWGSKQRNAPCSRCKPSVDGAGGVYD (SEQ ID NO: 13).

[0280] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14, which is the amino acid sequence of SzDa01 (Accession: G2GKN7; database: UniProt): MYVDTTLPKIRYLAHVLVTLTIVVMVALMVSLIPGVNVAQATEQKSDPPGLRNEKSIPG EVFTPKQRERPNGADRDWKASEASWPAGKRTAPIAAAPAGGRSASVGTSPVSVYQASG ARTASGADAGLVDIEVLDRKAAEKAGVNGVLFTVTPTVPATAGRAGSATAVAPSGKAR VTFDYAKFEGNFGANWGSRLELIDTATGKSVTRSNDSSARQLTGEINLPEARAGKAEPL ALAAVAQAAGSGGDYSATSLSPSGSWMGGSSSGGFSWTYPVDAPAVPGGPEPEVALGY SSQAIDGRTASTNAQSSWIAEGWDYEPGFIERRYRSCADDQGTGANNPGQAGRTGDLCF GSDQMVMSLDGSSTELVKDDTSGTWRPADDDGSKLERKTGADNGARGGEYWVLTTT DGTQYHFGLNKLPGAAAQRTDSTLTVPVAGNQAGEPCYASAFADSFCDQGWRWMLD YVVDPRGDAMSLWWAKESNYYGGNNKPAGMKYDRGAYLKNIDYGQRADKLFTQSA AARIAFTVAERCIPGSGFTCAEADRTPANAKYWPDTPIDQQCNAGEQCKDRNSPTFWST KRLTKITTQVNNGGLRDVDSFALDQSFPATGDGTSPALWLKSVERTGYGQDGTTATLPKAttorney Docket No. GH0197WO VTFRGEQMDNRVDGLEGLEPFSRYRVNAVDTENGGTVGVTYSPQECRRGGTMPAGPD TNTLRCYPQNWTPKGALEPITDYFHKYLVTQVREEDNVTDAVARVTSYEYLGGAAWA YDDSENTEARRRTWSQYRGYERVRTRLGSGTDVRQLTENRYFRGMHGDKLPSGTRSVE IEDSEGGKITDYPQYQGQLREEITYASDGGAIAAATKLTPAAVKTAERERAGTTPLQAY MVSPVTSETREHVKDSTWRRSSETTTYDSDGLPTQIEETTGSGRKACTRIEYARNTAQHI LDTESREVTTTGACGAPGGKVVDDTRSLYDGGAYKAAPTQGLVTEVQELNAAGDGYIT VDRTEYDIHGREIRTFDVEGNETRTAFTPATGAVPTKQVDTDPLGQTETTEFDHRGLPTA ELDANGKRVDMRYDPLGRLLKVWDIDRNQATQSASAEYDYTIRSDGPTVVTSKTLKGD GSYATSYEILDGLLRTRQTQDAAVGGEGRIVGDTFYDSAGRAYKSNDGYFNEQNAAPS LLSVGDNAVPSQTRIDFNGLGEETAEVTYSYGDEKLRSTTVKDGDAVTVIPPKGETVTTT YQDADGRDTKLREYTEADRSAWQDTVYSYDDLDQLTKVTEPGGGEYTFTYDARGRQT SATDPDGGTTRFEYDRGDNVTATVNALGKKLVTTFDKAGRATSLREDSADGPKRAEWT YDTLGKGLPVASVRYDKDGNAYRDEVTEYDANYRPRTTKTTIPAAAGKVAGEYTYTYS YTSTGNLAWVELPKVGPMAKERLVFRYNRDDLPISLSGAANYVTDITYSKYGEILRSEA GPAGRKLFSTYFYDDFTRRTDRVVHDRSVQPGRIDDTTYAYDEAGNVTAISRTPGEGMP DAQYGTDTQCFAYDQLRRMTSAWTAGEKCAPDGPSKETVGGPEPYWQSYEFDASGNR TKLVEHDPAGNVAEDITRTYSYGGDAGGPNRLREITSAGADGTTKTTFGYDKAGNTTTR QNGGTTQSLSWDIEGNLESVTEPRAGGGEKKTSYFYDASGERLLRTGSDGATTLYLGET ELTVRADGTESVERSYAHPDGATTVRSSDGGREVLLADQQGSTQTSVDMTEAGMPVTR RLLTPFGEDRGAAPSTWPGQRGFVGGTRDADTGLTQLGARPYDPATGRFLSVDPMVDY QQPATINPYAYANNAPATFSDPDGLFFPVLVGIAARIAIQAAIRAAARRAAQIAARKAAE ALRKRILAEARKRAIAAARKKAVAKAKREAARKAAAAKRAAAQRAAQRAAAKRAVA RQAAARAKQRAQRAAAQRAQAARAAARKARPKPRPKPKSQPRPRTKPSQKAKPAQQR KASPKQSVGSRAREEIRSEARGQAEDAVAEQASCRTNSFVPGTLVVMADGTRKRIEDVK LGDKVLATDPKSGRTTAQPVVSTILGDGTKNLVKITIRTGEASPENIRDAARPGGETDGG VPVDTGLLQAQPGDGDGNGATTGTGTGTVVATDGHPFWVPALGKWVDAGELEPGQW LQTSSGSWLQITAVQAWTQNATVRNLTVADTNTYYALAGTTPVLVHNCGGAQSLADR ASEIHAQAGSEIAMSKSTVAVVSAQTPKGVVNVVAGSGRGLNRAQKDMLSGVEFPARN IPGAHAEQNALLFINEMGWSPIAGGASRSVCSEVCAPLIRAGGGRITGRVYQNESGTKIR TFEW (SEQ ID NO: 14).

[0281] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceAttorney Docket No. GH0197WO identity to SEQ ID NO: 15, which is the amino acid sequence of SpDa01 (Accession: D6X696; database: UniProt): MAPFLPVAPNVVLLDMLRLIFVTTRSRKASVERGETMFKRRTRRALVISCVVAVAATGV PQPAFAAPSGDGGTGIVRGLKNFFGVDDDKPAAEPPELPDTAVPSNEQIPKGRAAPAPKR VKELTDRRTPDARYWQLSDGRVQSEVSAVPTHYEAGKAWQAIDPTVKPTDRKGFTLAN TTNVARTYFGERPGELVRFELDEGHWAAIGVKGVTGSLKPQADGSTVTYKNALGAGTD LTYTVGNGTVKEGIVLDAAPRTAPSYEFTLTTGGLQAVRRAGGSIALYSESSGPTRPALV IPAPFMTDAKKDGESPYEAAGTDRVEQKLSGKGTSYTLTLRPDAKWLSAPERRYPVVV DPTISIAPTSSQSQDVMISSDGPASNYDGNWRLSVGNTGSGSSRALLRFPLGAVPAGTKL DSADLKLYFDQTHTTGDSEVRLEAHRATAAWDETTATWNSASGITGELSGTSVLVDDR DSGTTAAKGSWPASGNTAFTQYAVNQDYLYNKDSVAGDTYTWQPNLPEDGDYQVEA HYVPASDRAANAPYTVTYSGGSKTYAVDQRAGTGGVWKTLGTHPFKAGTAGKIVLGD GPASTSTAVLADAVRFTKGGVVTKRPGESNTWHSFPVTRTVQSWIDGTHTNHGFVVKA GDESAGGPKGGPRYEAAEYAYKGEVANFPKLVLTWGRPGATLEAPQVIHATGAELKW SSYQDQDPISDRDDIVEYQVHRSVFQTFTPSPQTLIAPVPEGTTSYTDTTATPTPADSVDA FGNVFYYMVAVKTKDGQVVPAPTRIVKLPKAGRTTLVLQGDAADTTLSTTQPATGHDA LEDSGIGRPWLSVGNNSSTYGTTRALLKFPALSQVPSASRVIDAQLDLWGTTTTTGSDG AVYEVHALNKAFAEADASWNKANATTAWTTPGGDMDPAVADTVGTVTNDPARQSW NVTSLAQSWVSDPASDKGLAVRLKDESSTGPQERTIFLSSEAEEPQLRPQLVVTYLAKST DSTYFSPYTPARMTPGDRYTVDVTVTNTTTSAWKAADQALSYTWSLPDGTDATTGGN QLFTALPEDLLPGQSATVKAALQAPINSDSGNKRLDYVLKWDLRNKTAGTWLSATESIP PLAQNVRVEDPTSNQLGLEKFYSYAGKNTGAGSSMMTNLYAGNTVWQYNAFSNPGRG LTTFARFAYNSQDTSDTVLGHGWSAQAAMPLRLGTALDFHPNPNPTEVTLPDGDGTEH TFRKQEDGTWKAPAGVHYLLRQGEGVDCTPNKDGDPKAWSLTRPDRSQFFFDCDGYL TSITDKNGNTQTYTYEERKSNNKPVKFLRYITDPAGRQSLTVEYYTKADTSSPKIIDHVR SMTDISGRKVTFEYSDKGLMTKLTDGAGSSQPKVFGFVYDAEQGNKNAKLVKVTDPRG NATSLDYYYPSEGDDPKFHWNTQTITDRLGGTTGYVYRDPDATAGSFIETKVTDAENHT TTQSLDGYGRPNTITNAKSETTKLAWDADNNVTRLEEANGAFSTWVYDQKTGYPLEQK DAEANKNGTAAQTFTYSTGLNGHIADLFRKKSPEGRTWQFGYDTRGNVTSVTDPKGVA SATEGDYTSRSEYDTYGQLIKAIDANGHATANSDFQPVGYPGTITDAGGHTSAFRYDVR GNVTTVTNAKGAEVTQTYDAFGRPLEKKEPKDQAAGDFIVTPAPVYDANDNITKVTAP NGAVSTAGYDAADQVTESTVPGDLATDAPRRTTTTYDKVGNVLTTTSPKGNETATVGDAttorney Docket No. GH0197WO HTTTTTYDAIYQPVAVVDANGGRATSEYDNVGNIVKETDPRTNATPDPDDYSTKYEYD LNHRVTRTTDALGWTTSGRYDKDGLAIGRTDAEGNESLATYDERGALVESRVPVSKDA GGTIKYHTTRYEYDPVGNKTRTITPRGVATTDDTDDFASETVYDELNRVKEERSPFDKD TPGYTTPDTTVYAYDEVGNLESVSAPPSDGQTVRNTTRYTYYDNGWARTSTDPWDITT SYDYNQLGQQTKNTITSAGGSQQRTLSWDFYRSGNERAFFDNGVPVGRQAVLVDSSDF HNTATQGTWTRGQAEQQYGHDIASHPAGTGTAQFVWQLNIPQNGTYEVFVRHPKVSG AATDATFAVTHDGGTTSRSVDQTKDPGTWISLGKHAFVEDGPQKVTLTDKANGTVLAD AVKLVRDNTGESDTEQKDFTYAYDANANKVEVKDNSPGAKIDTYRIAYDGINRVSKVE ELAAGAVKNTSALTYDENGNPLTSTHDLTWSKIEYDARGLVAKVTNADSPTAGNQQIT TLTYTGRGQLLRQTKPNGNTLDLTYFLDGSVKQSVEKTSGGTVVSQHDLEYSPNGHRS KDVLKLMNADDSTAYINNTYTFDYDPQDRVTKVGKAGDSPSTETYSYDANSNITEQSV AGVTTTHRYDRNRLLSSTTAGVASTYNYDPLGRLDTTSTGGQSLEKYYYDGFDRTVKT RSGTGAAATTTNYVFDPFDRTVSQSTSGTDPKTTAFTYLGLDSTLLREEVDGKADKSYQ YLASGKRATQIKHKDDGAKEYSQYTTSPRGDVEAITKEDGKTRSTYGYTAYGSDDESQ MTGEDKPDAQNPDKESYNAFRYNSSRWDKASGTYDMGFRNYDPGLNRFLTRDSYGGA LADMSLATDPFTGNRYAFAGGNPISFVELDGHLFGLSLSDIGHAALDVVGLIPVVGEVA DVANGIWYAAEGNYVDAALSMSSAIPLVGYGASAVKAGRYAKKGMDALDSANDARK TTDRAGDAADAGNDVRKADGDAPSTPAKTDAKTCKVNSFVPGTQVVLADGSSKAIED LREGDNVLATDVESGETRGRQVTDTRSHEGDKHLVTLTVDVDGTEGHATGEITSTAGH HYWLPDSGRWAKAEQLEPGMWLRTSSGTWVQITAVDHDHAKKRVHNLTVAADHDYY VVAGGRSLLVHNECGPDYDQAASDKAGALHENLKVPGKKDGGYAYKNDTTAVVMAR DQAGNIRTIVASNRRYVPKAIKSKLDESAGEVFANGKGHAEMTALQYMDSQGWEFLGG AANRNVCAYCENAVRGRGLKLSGPTFPGFVQTLAGRFQFFGERMFRR (SEQ ID NO: 15).

[0282] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, which is the amino acid sequence of AdDa01 (Accession: WP_203730777.1; database: GeneBank): MLVSSRFRLLCHALIAAMIAAYLVSFPFTPASAAPVATAGAGPVNRPLPATAAANHGLP AGESAPPVPPAVATDGSKPRVPTAAEKAKRKLSAALAPTTKPIARGGFLLGDTSLVVYW DAEVNTGDPTSWGRWFATVTDTETGAEQRSIELGQADLSRCASPALLCRSFGAADGWV LDPARTYTVTVTEIAADGTEQTSPPSDAAHPRKVDDVPTVAAGQVVGCGCSTVLGPTVT AQALRGATVNTATGQYLRVERDFTMPSYGIPFNHARYYSSGNTNVGMFGPGWSSSYDIAttorney Docket No. GH0197WO RIVAADNGAVRVRAEDGSEAVYLARADGSFDAPAGIRATLSRNGTGWKLVPPDQRVLA FDADGRLLSIKNARGLGITLAYATAGPLSTVTDASGRVVRFEFRTDLRLITKIILPDGRST QFDYQDGRLLKTQDPRGYVTSYSYDAGGRLAEITDARGAKLIRNTYDANGRVTQQLDP EGGKTTFDWKPTEQIAVTVDPDGVTVSDGYKNNVLLYSQNGNSDTVNHRYTGKLNKN LVIDPKGNQEETKFDDAGNPISRTAPDPFLFTEVNTFDDRSNVTAVKDGRGNTWDYRYN QFNEMISQKDPKQGTGYEYTYDDKGQVSTRKDPRGKITEYEYDAYGDRTADVAPTGRR TEMFYDGTGRMTSVVDPRGTVAGASKDTYRTRFTYDQMNQVTETWQPGKGQPSRTTY DELGRVVVSTDPLSNSSRYTYDLASRLVEVKDPIGNVTATTWSAGGRRTSETDGEGNRT SWTYDNNGRVSTETSPRGNVTGADKALYTSVFHYDFNGNLIRTDRPYGTDGRRVEVDT AFDSLDRPTEQGDQFKNGTKVGYDNNGNVVSMTNERGEKLENFYDEANRRTGSSGGS GAAAVEYDDAGNVIKQTSPTGGVSTTSYDDDGRVVAVVEPRGNATGADPADFTTRYG YDLAGNPATETDALGNVAAAGYDPNNRVTSSTDANGNVTKYTYDAADRLASVIGPDA TGGQALTYGYNADGQVIKRTDPLGHTASLEYDRAGRTTVSTDGIGRRREYVYDADSNL VEEVTARIVELGRPDPNRPARTIFYAYDNLGRLISKQLGSGGTAYTYGYDAKNRLVSAA DPGGVLSYDYDVTDRLTTVTRGGEVFKYDYDSTDRIIGRTYPDGTKIGADYDDGDRITS LTATRGGTSANYGFHYDAADNLTGIDYPSATGITESRSYDRAGRMTRIAATKGDTTLSA FDLTLDKVGNPTKIIKATGEPGQPTITEAAAYTYDKANRITAECYGAQTCAGATAARTD YTYDLVGNRTTMKKVATGENTTTSYAYDAADQLTTETVSGTRSSSRAFTYDLEGNQTQ AGTDRFTYALDHTMTSASVNGTTTNYAYDAQGNRVSGTSGTGSSAITQTWSWDVNAS RPLLAQERQTGAAARDYLYDASGLPLGLIAADGTHSYLRDWLGGVSGVVSPAGAAEW EYAYDAFGVDRGTSKKADDAPDNPYQYAGTYQDTSQGDRYAMGVRSYDPSTGRFASA DPATQPATDQAVSTYSYTNARPTVLTDPSGADPDGMADTYADYYQAMACANYPPACA ATTDANTAAAGEGEQQVENPDWQNSKKLVDEAGSFVSKIGDEIVNLILDLVGFNDAKA CITEGDILACINTALQAVPWGKMFKAAKVAIKAIGVGRRLVDAYSRLKSAKNALAAIPR YIKKLTQTAEEAADSKKYADEAAKAAKGAKDAGGKAKATSQKAADNAKKAKKKEST TGESCEVAKGGTTRLASNSFAAGTPVLMADGSRKPIEKVQAGDTVMATEPSAALTAPS QVIATVAGTGEKSLVDLTLVGAGAGGGGPPAVITATTGHKFYSLDRDQWVPAGELKLG ETLRDNRGGKITLTGLSEHTAETTVYNLTVDDEHTYYVVAGDNPVLVHNCGAGADAES LAEAARYRADELHKALDPIARSKRTTAVISAYDHEGQIVDVVSASGRGLSRAQKELLGA GEYIAKSTGKRSHAEMNALAFIESKGWTPIAGGASRNVCGKCSAGLRASGARLFGGTFP GRGSRMRLFRWF (SEQ ID NO: 16).Attorney Docket No. GH0197WO

[0283] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17, which is the amino acid sequence of MGYPDa23 (Accession: MGYP000056170311; database: MGnify): QEDNTTQEVITWLFEGFVPVAKIVDDQKYSIVSDYLGKPFQAYDSEGELVWAAEHNIYG KVKTLKGDKTFIPFRQLGQYEDIEIDLYYNRFRYYDCHTGTYISQDPIGLAGNNPNIYAY VFDSNTEVDVFGLEVIFDETLANMARQAHDVLREKDKTKIKFNHSTVAIAEVEINGEKQ LFASGNGAVLSPAQRDKLKELGIPEENIFSGKRFKKILEDLEATKLANHAERVIIRNVPEG AIFTGKWGISWAGNQRNESCVNCLSFVEKVSNKHH (SEQ ID NO: 17).

[0284] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18, which is the amino acid sequence of WWTPDa07 (Accession: tig00001818-10-11279730_497; database: GeneBank): VRSGEWDAAADQDSSGNFTEFTLPIEAYVSDFQVGWLLQPDINVAVYLPITSISGHSLTV SGSYGSLAPSSIGTHFRIHAPPGVERTGPFFSGFEAGTLSRFTSASSSRNLFGGYRYESPLA GVRDDLWMAVQGRQSGKNFTGLHYTLHRHYDPHLMRFTSTDPAAAPFFNLYAYCQGN PSRFFDPDGLDINTNATGCRLSPEDEEYFARSAGRTAALMDAMNCFGLFDRTKAAMRD QKSFSEVAGTFSNEVATTDWRTVTTSHFYNGANTLSGGWMGEVLDAETVAGWTGAKV NSREWRAGAASGSFAATTAAMLNPYSAMTVGLLTMTHGGYLAARIAAGDDSIDPFQA NAEMFGGAAMFGGGYVRLPSGSKGPKWKGDLYETPNLSVRARVRQLRESMLPDAANR VTLGAGILEDAQGVRRMVIATSEKRPYLRAGVRRNPGETMVPIVGKLHAEKKITLHAW ATRSKLVSVGATRPICGGCQDAILPTNASMATPLKRRRH (SEQ ID NO: 18).

[0285] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19, which is the amino acid sequence of PdDa01 (Accession: JH605467.1; database: GeneBank): MIDPNSSETEYKYDKLYRLIEQIDGESGKTKYRYDKVGNVRFVTDPRDKVTEYQYDRV YRLQKEIDANDETREYGYDLAGNLTSIQDRRDNTTIFEYDDVNRQTSRINPYGARFKIDY DKVGNVIKETDELGRSTKYDYDELNRLEVVTNAEDGTVVYGYDKVGNVISVQNERGKI TRYEYDEINRQIKITNALGFETIIDYTNDTDSNQLVVLVTEQVDADSSRSSEWRTDSRERL QTFTNADDTTTTYSYDGNNNLLTVVDELGRQTAYTYDELNREIRVTDAEGRTVETDYD ANGNVVKVVDELGYTVEYGYDQLNRLTSELDNLNRSLTYSYDSEGNQTKVTDSNGGEAttorney Docket No. GH0197WO TKYKYDKLNRSLEITDAEGGKTTYTYDGVGNLKSVNDPNSNNGGNSNENGHTVTYSYD ARDRLIKEIDALNQETTYNYDEVGNLTSVSDRSGNQTIYKYDEINRQIERIDADGNSFKT EYDKVGNVTKETDELGRITTYSYDELNRLLEVTYPAIDGVVSSSSSTYDAVGNLLTSTDE LGRTTEYKYDKINRNTEIKTPLGFITKFDYLDDTDNNLLVLTVTDAVGRETVSRTDGRG RLIEFVNADVSNVTYSYDGNDNLLTVVDELGRATAYTYDELNRETRVTTPLGNTTTTEY DSNSNVVKITDHLNNATRYTYNELDLLATETDARGFVTTYTYDEEENLASVQDPLDNV TTYTYDNLNRLFQETAVGVGTRTYSYDEVSNLTQLLDRNGRIVQFEYDDWNRQTAELW LNGDRIVNTIAYDYDAASQLVEVSDAVAKYSYKYDDDSRLTKEIRDTFGFTDGVVLDY THDGVGNVLTVTDTVGGIQLSTETYTYDAQDRVISISQTGNGVTPKTAVVTYDDAGQLE QLEVLNAFTTAQTFDLDGRPVTRTHTTAVGEVLAYGYEFDSLNRITAITSPNGESSFSYD STDQLTSASSDFQEDENYDYDETGNRLGNEIGPHNRLLNDGVYSYEYDDEGNRISRTEIA TGVVTDYEWDWRNRLVEVSSSNELGEVLSSSQYQYDAFDRRVAKLVDSNGDGIYELVE GFVYNGDSILLVLNGDEVNQRYFYGPGIDWVLAEETAGEGVEYALTNHLNSVEFILDSA GEVINEIVYDSFGGIVSETNPGVDVRYGFTGRDFDKETGLGYNRTRYYDFVTGTFLSVD RLGFEAGDVNLYRYVGNSPTIYVDPSGMFWEGFAEFIDENVNKPVGQFIADNPLVEDAV RIADSVAVGVSQTANLVGFDIREFREDIYGEGVNDNLNEAVVTASTVATLALGGYALSG LAAGSLGKVAATSINTGLFNTGFGLTTQLLFNGGDSRQLDYNSLRLDFAFGALGARLGF GVTARVGGGFATQFAVSTAFEGTFAGFEQVGRNLISGERLTQGVGENILSSILFQGFSEA LSNPRQALQAGRRLLRGADNLADDFLGVVRGPQLATVDGVDSRLLRSVDEVPNVPKGG DNVFASIDDPKSRGELTKLIQQNGNVVDLSDRLIDVATSISNRSLHELAKDVHKLKGDKR AITQSAVAIVEARLVNGKKQLFASGSNATLSPRQRELLKQFGVPEENIFSGSKFRSSDKL KNHAERVILRHLPYRAKVLRWGISWASKHRNIPCPNCYPFVEKAGGIIE (SEQ ID NO: 19).

[0286] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20, which is the amino acid sequence of MGYPDa25 (Accession: MGYP001677015708; database: MGnify): MVQSVHNLAGGLKTLDARAIEKSTVAILEVELRNGSKTLYAAGSSGWLNPRQRALLEK LGVPKENILSGKKYTLSKDVVKNINHVEQIILRNIPDNTKVTRKGISWGSKQRNAHCSRC KPSVDGAGGVYD (SEQ ID NO: 20).

[0287] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceAttorney Docket No. GH0197WO identity to SEQ ID NO: 21, which is the amino acid sequence of MGYPDa26 (Accession: MGYP003599500301; database: MGnify): GGEGHPASKTIAARASQTGATGQNHSKPAANDAEALYWASLNQQAEQKAQALAIEGW GTGTDGPQQQTAQAKIAEVYYFHTDQVGLPEELSNAQGQLVWQATYKTWGNTQAEE WKLVEADGSKTHPLDAGDQPESEAKHQNLRFQGQYLDRDTGLHYNTFRFYDPDIGRFIS PDPIGLEGGINLGSYSPNPIGWIDPWGWSCGELSSKARQVHNMAGGKVPDARSIRNSTV AIFEVKLPNGKTTLYAAGSSGKLNPRQRAELVKLGVPESNILHGKGVNLPKDGNPVNAN HAEQIIIRGIPDGSSVTRKGISWGGKQTNAPCIHCKPSVDGIGGIYD (SEQ ID NO: 21).

[0288] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22, which is the amino acid sequence of AshDa01 (Accession: A0A7H8HBT9; database: UniProt): MRYHHVRVTDPGVTGGTVPGVALYAKKITYTGQGKIEGRYSITFTRDRDRGEPRRPDV VIDARNGFKQVTADLLRRVDILLDDKPIRAYELNYRTGAFAKTLLQSVTQFGEDGSPFTT HTFDYYDDIRSADGQYQAFGPAAGWSVPGDSLGESVPDGHASALSATTSRSTGGHLYV GFNPAVVSKSGSAGAKVGFNAGTSEGLLSLTDVNGDSLPDKVFRTGAGVFYRPSLSGPG GAPKFGDTPIRLPGLPGISEESTRSTTSGAEAYFGVAAQLDHVSTTTRTDRYLTDVNGDG IADLVTGGSVLFGHLDAQGNPVYSANSNDTPVPIGGGTVNGTIVGDQTAEIERQVDAFPL LDSVRRWVAPYDGTVRIAGGAKLTSDPGDDRAADGVRVAVQQEDTELWAQRITARDH NTYVPSGVDSVAVHRGDRLYFRVQSVADGRFDQVNWDPDVTYTGVTPATDVNSLDVY RYLASRDFTLGGRPSTVTAPVTGTLHLSGDVSKVGPTTDDVAVVITRNGTDVFSRTLPA ASATTVPIDLDIPVSAGDTLSWRLKVDSPIDAGTLHWVPSAHYTAAEGGVPVVDQQGNP TLVIDPPFDLDLYPVDTATAPQGTYTADRTGDLVVEPNLALAGDAHVVFTVKRRGALL AKRVIDVAGGQVPAPESLRVTVPVTAGDELFFDFSTLDPGLPAKVTGQSASVSTDGTTFT PVPSALHAAAEQGAFAAPYRGWAAIGYQGNRDRATAPIKQSDLVIDASYRDKIPGAPKE SDVPGFDGRVSKPNAVVLAPRPALERWGSDDGNTWVGAGSAASSRWGTDRIDVAQDA DIAGATGVSRVARIQQISTTFGASIPGVPIGAGASVARGRSSGQVDFLDLNGDRFPDVVG PAGIQYSDVTGGLGATRGSLGTGSVRESDSTAFTASANAGSPARTEPNGRGMSAPAADK PATTAQSGSELPSLGIGGSVGGGQSGAAFDLLDINGDTLPDRVYSDGTAALNLGYRFAA PEPWPGGPLADSNSRSSGVDLGFNTDFYGLAGGVSATSGQAKTEAGLQDVTGDGLPDR VFSRDGQPLSVAINTGSGFTAPTPLGGSQAGITTDTNAGLGAGAYFTFGFCLGILAGACV VFNPGADTSRGIGRSEVALRDVNGDGYADQVRSTRDDELVVAENRTGRTNLLRSVSRPAttorney Docket No. GH0197WO LGARFELDYTRDGNTENLPQSRWVLSRTTLFDGHPGDGQDFQVSTFHYADGRFDRLER QFLGYGRVVSEQRDGDAVYRSTTSEYRTDSVYTRGLFTRTYTADGGGHLFTETLNSYV LRDVATGKVADPASTSATVFPLLAHTEQRFYEGGAAAGKTTATDNEYDQFGNLTRVLD TADTGTADDVETRMQYTAADAACRDRNIVGLPDVVDVVAAATKTTTRHRESTIDCATG EVRQVRAILADGSKAVTDMEYLGNGNLKAVVGPVNKAGQRYRLDYGYDPVVGVHIET IVDSYGYRTAITHNYKYGLPETTLDKNFQRVHTDYDRFGRVSFVAGPYEIPDNRVTVSF EYHPEAPVPYAVSRHIDTTAGGVRDDTIDTVQFTDGLGRVLQTKKDASVAPAPGVTPEA VMTVSGHTVYDFAGRAVEQYYPVTEPKGDKNYVFNPAVDSVAPTRLGYDILDRTTSTV IPDGSVSSTSFGFGADRSGTTRFETVSTDANGKQRRSYSDTRQLTTAVLEANPAGSQPVI WTSYDYDAMGQITSVVDDRGNTTKAAYDNFGRRTVVDSPDSGKVESGYDLADNIVSK VTPTLRAQRKAIQYDYEFTRLTAIRYPVFSGNNVTYTYGALGAPENGAGRITSIHDAAGT VTRGYGPLGELTRETRTLGKDVGTWEDRGQSFTTTTRYDTWNRVLETTYPDGEELTYG YDTGGQVTTATGVKNGNQYKYLARMDYDKFNQKVLQETGNGVRTTYAYNDTDRRLA TLKSDQPDGREFQNLSYGYDKVGNVTTLTNDVAVPGAKELGGPSTQTYGYDDLYRLTS ATGSYRNDNDKLNHYQLDLTYDTLNNTTGKTQKHDLTTTSGVTQPQYNTSYTQKYTYS ALPHAPSKIGTDTQKYDAGGNLIAITAGRSTDAVVLAAADTSGGKRTQYVWDEENRLE CAFDDESSTVDQDPASCDGRHEQPTVRFVYDDAGNRVLKDGEETHVSIGTTYSRVDGK AVKHYFVGDTRLLSKKASDSGYEHDQFYFHPDQLGSATYVTDERGKLASHQEYFAYGE SWVEETRARKPVPYQYNGKEFDKETGFYFYGARYYNPRTDLWQSADPATGSYLDGTP NGGVTQPFNLSSYSYASDNPVRLADPDGLSTWNRVMGGLKLVGGFSEAATGAAFGTAT SWTGVGAVAGAVVFVHGADTAATGLRQLWTGEDESSLTSLAIQHTTGLSKETVDRGEA VVALGASLGMSAATKAPQVLGATAGGGGQTTRAVGSAGLIGGGLSQPGALRAAAVEV HDLALLEKGGEIRHTMSTVALHEARLADGTTQIFAAGSSGRLSAAQIEKLVELGVPRANI VRGGLHAERNILAAVPEGAEQVRWGIAWGARNKPIPCDKCAPFLKGVLEGAE (SEQ ID NO: 22).

[0289] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23, which is the amino acid sequence of MGYPDa21 (Accession: MGYP001278432191; database: MGnify): DLIEQLAERLGNHANLTDELIAAELQAMGVAATDDIVARVWSGLNASALRQSGALRAV ALEVHNLAGSQIAINQSAIAVVEVTLADGSRQVFASGSGGRLTTEQVRRLIALGVPPENILAttorney Docket No. GH0197WO FGAAFTNGFTQAENHAERILLRNLPQGAVVNRWGISWAGSHRPIPCDVCRPFVAQAGGI LEGAQ (SEQ ID NO: 23).

[0290] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24, which is the amino acid sequence of PpDa03 (Accession: A0A3S0YAN6; database: UniProt): MAGTETHYGYDEDGNCVTVRNGEGEIRHFLHDGRGLLIRETAPDDILHYRYDAAGRLT EVTSATSHIQLAYDKRDRVLEEHNSGSVIRRHYQDASHTVTRSLLWEGEEDSAALTSSF CYSATGELRQVQLPDGAELMLAHDAAGRESIRHSNGGFTQQREYDAMGWLTREMSGQ QQDGRLHASQTREYLYDGAGNLAGVRHNRETWGYRLDATGRVLSVLSGGAGRAVET DEAYRYTRNGLPQDTTRLTEWQAGRLTQHDDTHYQYDKAGRLIRKQVIQPGYRPQVW HYRWDSRNQLRVVDTPTGERWFYRYDPFGRRTGKRCEQTQDDVRYLWDGDQIAEVR HYRNGERVARRHWVHNGWELLVQQRQNTDGRWETDFVTSSQNGEPQALYRPDGTLC WQAPKSTLWGQRPGSTEDPADPGLAFAGQYRDTESGLCYNRFRYYDPTGGCYVSPDPI SILGGESNYGYVQNPLGWVDPFGLASCEHLNKMAKEVHDLAGGLKTPDARAIRNSTVSI VEATIDGKKVLYAAGSGGRLNPRQVEALERLGVARGNIFKGARVTNDFTKLENHAERII LRNLPDGAVVNRWGISWGGLQRGIPCDHCAPFVRDAGGFF (SEQ ID NO: 24).

[0291] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25, which is the amino acid sequence of SbDa01 (Accession: A0A7G6KCF9; database: UniProt): MAQKVHDLAGGLHAPDLRAIRNSAVAIVEATVNGEKILFAAGSAGRLNPRQVALLKEY GVLEENIFRNSAVTKGFEQLENHAERIILRNLPEGATVERWGISWAGKQKNIPCPHCEPF VRDAGGFFDKIW (SEQ ID NO: 25).

[0292] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 26, which is the amino acid sequence of BlDa01 (Accession: A0A6P2WMK2; database: UniProt): MWNGILLVQETYHDRWGEEALTYLYESNSYVPLARIDQGKAAANDANARDAVYYFHN DVSGLPEELTNAEGELIWQARYKVWGNAVQEEWIARAPQQPVPEWGELQVATATSVH MPRPQNLRFQGQYLDRETGLHYNTFRFYDPDIGRFINPDPIGLLGGTNLYRYAANPLVWI DPWGWACGELSGKAQEVHNLAGGGDARSIRNSTVSIVEAKVNGKPQLFAAGSGGRLSPAttorney Docket No. GH0197WO AQRQALMKMGVPEENIFYGKKSIDGFDKLENHAERIILRSLPEGAEVSRWGISWGGLQR NASCPSCQPHVDQAGGIFD (SEQ ID NO: 26).

[0293] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 27, which is the amino acid sequence of PpDa04 (Accession: A0A5M9IK01; database: UniProt): MSDALWAARMGDALTHTSMMADILGGVLEVAANVAITALATAAVAAAIGVTVATAGI GGCILGAVVGIVVGMAMSKTGADKGLSKMCESFANALFPPVVEATIAVGSADTFVNSIP AARAAGSIPSHVAPAGTELELPPPEPDTAPQAEPGFLDMAEGFFSQLWRPTVATPAPGVV PALTDMVLCARHPPMPPQLLAEGSDKVTINGQPAVRSGDRSMCDATVVSSGLISPNVTI GGGTVVVQEIRSGKTPGVGLAIEVLMMLKGAKGKTLSDLPCMLLSGVTSYAVSQAMG AAANATMGSPNPVHAATGAKVLGGNTEMDFVLPGILPITWQRFYNSRDKRRNGMFGA GWSVSYEVCIEILPHPDGGEDLVYTDEQARRIHLGAMPLGTAMFSAGEGLSIRRHRNGQ VLIESEDGLYRLFDSAPGDSAFLRLSQLGDRNDNRIYLDYDERGQLVRLRDTFDLIQIELT RERNRVCQIVRVYPDHRREPLALYSFDAAGDLAHVSDATGQTQRRFAYDSSQRMVEHQ LPTGLRCFYEWALIDQMEWRVIRHWTDEGDEYRFEYDLQAGITRITDSLQRVSTRLWNS QHQIVSYTDNLGQTWLFEWNDERQLLSATDPKGGRYAFSYDDSGNLIGEHDPLGRSDST VWLPHWALPRFQIDAAGNSWTYQYDSRGNCIRETDPLGYSTQYRYDAHGQIVEIIDAAG KSKRMRWNQFGQLIEYIDCSGYPTRFSYDERGFLQTITDALGERSHFSVDAKGRLLTSQS PDGQMDYFQRNANGQLVSHTNAAGHTTHYRRNRRGQVSQRTDAHGRTVEFGYDAFG RLQALTNENGERYLFTWDAGDRLEKQQDLDGSAKYYAYDALDNLASVESVPALYGTG LSVVPPTRAKPIVHRLERDAIGRLTAKVTDDGRTEYVYDVLNQLLAVTFTDHQGNSQAL GFSYDALGQLLEEASPAGSLRHHYDELGNLTQTQLPDGRWLNRLFYGSGHLHQINLDG VIISDFERDRLHREVMRTQGQISTYLGYDRSGRLRSRQRRYTQQASLLPSAEQSNFEYDL ANNLVVKRDQHAKGQRHQLLHYDATGRIIASQGDIQGQAEIFAYDAAANLLDGSQQGA GLVVHNKLITYQDKRFRYDGFGRVIEKRSSQRGLQSFRYDAEHRLIEVRNHGTAGDSVI CMKYDPLGRRQEKCEWRNGFQTNKTTFVWDGLKLLEEQHNSQKSLYLYATSGYEPLA RVDGRGHQQKIRYYHNDVNGSPLQLTEADGSPVWEATYKVWGNTLQEVQEPYYIEGQ NLRYQGQYLDRETGLHYNTFRFYDPDVGRFTTRDPIGLAGGLNLYQYAPNPIGWIDPLG LSCSLSAKARKIHDVARDPFAKRNSTVAIAEVTINGKTTHYVAGSGGTLSPAQRKLAAE MGIPPENILSGTKVRSSNADPTQAKLENHAERVIIRNLPEGAKVEKWGVSWGGKQTNAS CSNCQPHVDASGGIFD (SEQ ID NO: 27).Attorney Docket No. GH0197WO

[0294] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, which is the amino acid sequence of CsDa01 (Accession: A0A3E1NUV0; database: UniProt): MLISNKHFVPVIGLDIHIVILFGFPIPLPHPYLGFVLDPMDYIPFLGASTKVNHVPRGVSDT SGIIIILFHFPMGGPWLLAPLIGHDSVNFYGAKNTLVEGRMLSPTGHMLMTCNDIGLPLSL KPGKKLIPMPGMYLPTSYSIPLSFGKPVIVGGPYVPDWAGVLMNLLMSFGFGALLKGIG KLGKKMMTKFNHALKGKLGSNKLSKFLCKHGFEPVDLVQGIVISEGLDFELSGPIPIVW ERTWNSDSAHKGLLGHGNHLFYDMRVQEFAAENATVVLLRDGRSAVFDALPYSGDKE YNRHEKMLLTRTDADEFVLYDYEDKLFYTFKKIRETDEQFRLFAIHNKMNFLVTLHYN QKGHLLRITDSAGRQLHIKNDFEGRITSVTAHHRGQEMLLVSYEYNEEGDLTAIADALG QATRISYRNHLMVSKTDRTGQTFYWEYDNKKRCIHTWGDGGLLEGFIEYHPSEGYNLV TNSLGETTTYYYSPDFVISQIRDPMGNSTFINYTEDFEIYQEIDEEGTVAGYTYDDMGNM TSVVQPDGSSYSFLYNEAGEMILGMDAEGNSSTYTYYPDNGLLHTNTRPDGSIQIMRYN EQLLLASVEDTDDNITTMEYDEDFNLVKMVLPDGGEATWTYDVWGQCTSSSNPARQV QRFRYDALGRITDVETPDGNHISMEYDAYFQVLRAVDKHHDVKFSYTPLGNLRTREEN GAKVNYIYDTECRLRTIINEKGESYQFVRNAKGESIRETGFDGISYGFVRDATGKVIKVE RPGQKHSTYEYDYNGMLVRAEHSDGTWSSFAYDRNGLMIEATNENGTVSFKRDAMGN VIEERQNGHVVSTQYDENSRRMRVQTSLGAQVNLLRNRMGDITGMRANDSISRDPWTL QIQYDLAGMETERLLPGGISSSFTYDKAGMPASQTINSNKRTIRKTYYRWDANQRLREM VNALNNSSVKFAHDNFGNLAWAQYEDGHFDYRQPDKTGNLFRSVSQNDRKYGPGGQL QETPDARFEYDAEGFLIKKISSGLKVWEYEWTGRGTLQQVTRPDGKTVRFEYDALGRRT AKIYGNEITRWVWDGMNPVHEWTYPVDERPQIRIDDMGNIMQQEEPVPAETLITWVFE GDSCIPVAKIINGKQYSILSDYLGVPFAAYDGSGGECWSCELDIYGKVRKLAGSKGFIPF RYQGQYEDVETGLYYNRFRYYSPDEGVYISKDPIGLDGGSRLYSFVNDPMSLFDLLGLH PIFDDALAEKARQAHNVLKRNIPGKPEIGFNQSTVSIGEGTLPSGEKQLFASGNGAKLTP AQRAKLIELGVPPENIYSGVAYMKKPVNKSKVKSVRKAYNLENHAERVIIRNAPKGTKF KKWGISWAKYQKNEPCSNCKPHVQCAS (SEQ ID NO: 28).

[0295] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29, which is the amino acid sequence of MGYPDa22 (Accession: MGYP001462196871; database: MGnify):Attorney Docket No. GH0197WO DEYVPPVGMPSPWLYGDTTNVSEEYIKKKFDRTFLLSKGFPVEFPDFLELLNSLGFAREA TEDEIIAHIYQYTHEYQALDYGYKVLRDRGEYSFIGSHSTPSEVWTGKEYSEEEVLQIGY ANIIYDEQQREIAKISLDLERNMEESRFADPEAKAVLDLGSTNPAYNFRALRFLIERLFNEI SLGVQQNQAILKNHLHRFPLLRMYVTNPLALPHLLGSSLPQNSWSRQIIDFDINFDEIIITD NISAENIPVNYSKLQTDVKSLEANIESTRDFAGFKAPLLDIYDEFHLEDSDSNPITAVAGG RTRTSANASAGLNSIELKLENTDPSQRKAALDELLMQNNEESWQALLMTYDSPHEDVR SKAQQTIKTRLKKDATFKKYVVNLVNTPQGLLSDFAFNLLTLYNSTEEINEDHYRKIVLQ NLHQANDLVGIFVSKVQLYFEYLHRNDAIDFQIDTTWINEGITNIEETYFHDLGSLGIRTT NFIEQLSIGLESIESMQEFIPTEISQRGKLKVEIHPQLINLITLSSKLGGEDAHEHFQSILTSL QAWPGKFAQAFSEDLYSTFTRVKAKLQSSLNSKALSSFKSAWATFKTDMITPLLTDTDV IILKLEQLKLTASSAPNKFFAELDTLSYQIESITEKAQYALSATDLMDTYSEFAWRNEVFK DEYLEEEQTIKNAWGLIALAAYNRDTDPEGARRQFNQVIESEVLKDVDEKIAELYKFQQ GELDTFMDIMILLVSIKTGGLAGGAARTGLGFLTRAGASTFARQATVAIATLGAEATAF HLTSRSLKTLANRGDFMDDTFWEDYLETFLMFGAMKGGGALFQKFLAPKAAKFMQST KFPNLAKATSFLGKQATVFSFFQAWTIGAHWIKTGEFISPTDKEFWQIAAQNAFFLGAIQ FGMAATRPLVMPKQSQVPKEWLETNNAEGGMLSRQMAAWKSNKQASNAEAMEIMKQ AKSILERRLGIFERIHANYPAELSKVEIIKIRSEINSLISSINNTLAFGNAKVKPSATHPGTY YYEGSFNQVAPSLREQGFVVAESTVPGQVNVVNSLTGEASTFLSLPSGSNPIPAGGISVA ERLNQLKAEGKSGGPRKQAIVENPDAYYFDLARKGGKGEYRLRPGLPKSPSSPGQIKFR TAKTALPRNLNEPLALYHLSRSVHNVAPKSMTTQGNITIEQSTVSIVKVKLADGTMTFY ASGNNAYLTPNQRVLLKLAGVPEKNILSGEEFRVNDKLENHAEKVILRNLPAGAVDLE WGISWAGGQKAASCSHCATVVDQTKIQRHRP (SEQ ID NO: 29).

[0296] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, which is the amino acid sequence of FlDa01 (Accession: A0A0Q4RZG0; database: UniProt): MVYDKPEPIKNLITWVYEGGSFVPSAKIIGEHKFSIINDYIGRPIQVYNEVGDVVWETDY DIYGGLRNLKGDKSFIPFRQLGQYEDVETGLYYNRHRYYNPESGGYISQDPIGLLGGSAS YKYVHDCNNCVDIFGLNPVIFSEELSKIAQEAHNVLLEPGKSPRGFNNSTVSVAKVDVN GVSTLYASGNGASLSPAQRTKLVELGVPEENIFSGKRFKEIIDGDTGTLTKLSNHAERVIE RNIPKDASIKEWGISWASKQKNEMCNNCKTHFGCK (SEQ ID NO: 30).Attorney Docket No. GH0197WO

[0297] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31, which is the amino acid sequence of MGYPDa24 (Accession: MGYP000620945751; database: MGnify): QETGFVEVSKGDIGVIGNAALARKIEPDPYLCPLRFQGQWEDEETGLYYNRFRYYEPMA GCYLSRDPMGIYGSYRPGAYVPNPALWIDPFGLQRQPASELSGCEELGEMARAVHDIID DPRAKANSTVEIFRGTDGQLYASGSGSRLRPAQREALMRMGIPASNIFSGVAFQGADKL ENHAERTILRNMPEGVKVQSFGVSWGSRQRNVCCTACAASMIPNLAN (SEQ ID NO: 31).

[0298] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, which is the amino acid sequence of AaDa02 (Accession: A0A2S8AG27; database: UniProt): MAREVHNLARDERALRSQTVSIAKVEKDGVSSLYASGSGASLNPRQREKLEELGVPKE NIFSGKKFKVFIDGERGIQTKLANHAERVIERNIPLDSEIKEFGISWSSKQKNEMCPNCKE HFGHSH (SEQ ID NO: 32).

[0299] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33, which is the amino acid sequence of MmgDa01 (Accession: JANFCG010307071; database: GeneBank): SSTPVGSLRSIAMAAHSKAATPTALEKTAVALAEVRLKNGSVEYWAAGSGESLSGIQRN YLESLGFRVISGKAGFHAEEQIGYLLPEGSDVLRWGISWTTPQKGIPCIRCNPLLDTIGGVI EK (SEQ ID NO: 33).

[0300] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, which is the amino acid sequence of PbDa01 (Accession: MCL1918637.1; database: GeneBank): YQNDPNGMPLRLLDENGIIKWEGHYSAFGLVDRVSVEAVGQPLRLQGQYFDDESGLCY NRHRYYDAVVGCFISSDPIGLDGGLNPYRFAPNVLGWIDPWGLSCAVLSARLARIARIV HNLSGNPRAIRQSTVAIARVRINGKYQLFAAGSGGRLNPAQRAELVRLGVPEGNIFHGR GVTNGFSQIENHAERIVLRNIPEGSSVVHWGISWGGLQRNLPCVSCRPYVLGT (SEQ ID NO: 34).Attorney Docket No. GH0197WO

[0301] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35, which is the amino acid sequence of BcDa02 (Accession: KVH32961.1; database: GeneBank): MSSDHPGGYVEHNRLKMFEDKRFEYDVYGRLVRKLSGHGPAKELVLEYDDWNQLKA VVRKDRLGIGTTHFEYDAFGRRIRKFNGSYASTDFRWGGMRLVQETYHDRQGEEALTY LYEANSYVPLARIDQGKPAANDADARDAVYYFHNDVSGLPEELTDAGGELVWQARYK VWGNVVQEEWIAPVRHQPALAWGEVRAVIESPDHVPRPQNLRFQGQYLDRETGLHYN TFRFYDPDIGRFISPDPIGLNGGRNLYRYTPNPLVWIDPWGWACGELSAKAREVHSLAR DDFALRNSTVAIVEAKVGDKTELFASASGGKVTPAQREALERLGVPKENIFWGAKAVN GYERIENHAERIILRNLPDGSEVTRWGISWGGLQRNASCIYCQPHVDEAGGIFD (SEQ ID NO: 35).

[0302] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36, which is the amino acid sequence of LsfDa01 (Accession: UHQ21442.1; database: GeneBank): MTTAAKHFDPQLGIDIHMYVFPPVPLPVPLPTPHIGIVLDPFDYLPFLGGTVHVNGIKRAT AGTGGLNLHIPMGAYHPAFLPKLPTGPQTDDELFMGSMTVSADGDPFSKLAMPVLDCN VVGMVPPFRLRKPKKPKLSLTLPTAVNLAIPTNVNVGGPPTISLMAMAMKGLFKLLGPV FKRGGKAFKKLRQKVFGNMKPGFLKCKVLRAEPVDIRTGSVSVTHEDFVVPGRLPLAW TREYGSNNDHVGACGYGWETPADIRLELDADGSVLFHSGEGVAVFPQAPEAPGLAHAV REFVDGARLYAQGDEWRVRSKDGLRYAFARPAANLVALTTQTLPIQRIEDASDNAWRF ERRGADLVRIVESGTVDPVSGQPLQGRFLQATSRQGRIEALELHDPATGRNHPLVRYEY NDAGDLVSAFDPLDAPRTFDYRQHRMVRHTDRLGLSFHYAYDEQWRVVHSWGDGGL YDYRFAYDDLLGDVEITDSLGHTSLVKFDEYRLPLCEIDPLGGVTVFEYDEAGRTTAVV DPAGLRTEFVYDERGNLLQLTRADGSTLQVQYDEEDRPVAVIDPLQASWQQRWDERGL LVEQSSPLGARSTCSYDPHGQIIAYTDALGAVTALHYDRHGQVQAIVDPLGTRSSFQHD ALGRLLEQANAAQQRTGYQYDAKGRLLNVVQPGGTTVRAAYDAEDQLIRYTDEAGAV TQLAYVGIGQIARRLQPDGHLVQYEYDREEQLIAVINQRGERYELKRDALGRIVEEVDY WGQARHYRYDAVGRLTGTTDPLGQVIAYATDPLGRITAKTLPAAERPGQQIKEIFRYDP RGQLIELRNEHRQVKRTFDSEGRLVEEDQDGFVIANTFDALGNRIVRTASAGNRVACEF DLRGQLSRVTINDDAPILIERDTLGRALTERLSPQVRRQLRYDERGLLTAQAVLKDEAPLAttorney Docket No. GH0197WO FDTSYAYDAVGNLTQRHDSAHGADLYTYDPMGRILAHTDPRGKLTQFLNDPAGDRLRT RVHAKRQQQVVGGEPQPDEWTREGDYTGQHYVFDRAGNLVRRGDANSAGNDDERAL HLRWDANQRLLESTKDGATTRYGYDPLGRRVFKRNPTHTTWFYWDGDALLGEVSHDN DRIEVPALVNDGVVVDLFEARQRKRVFDGLHKRVREYVYYPSSFVPLALIDRQRDAAV NTSPAGTAAPEAATTTEVFHYHVDPNGCPTRLTDASGKVVWSVSYATWGEVTRVHVA EIDTPIRLQGQYSDVETGLHYNRHRLYMPELGDFASQDPLRFMAGENLYGIAPNTYAWI DPKGLSCQLSHIARQVHDLAGNHPKAKQMSTVAVVEAIVDGKKILYAAGSGARLSQMQ RELLEEYGVPASNIFHGAAVRANDQLANHAERVILRNLPPGAKVTRWGISWAGNQKNA SCPNCQPFVDAAGGVLD (SEQ ID NO: 36).

[0303] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37, which is the amino acid sequence of SmgDa01 (Accession: JAHZIM010172802.1; database: GeneBank): VTSVVGGIAMVNGADDFLTGIRQMFTGRAERGATETFVTEAARLVPNLTRDQRENIGTG VSMGLGFISPVPGSNAPRMALAVAGGGQRMASGVTRAARIAEQARAAQIAANGVRVA MSTQDAPSSSTPDAPSSSTPDAPSSSTPDAPSSSTPDAPSSSTPDAPNSTSSSSAVARTPEQS AQAQLLPRSDIDYRLQPGGALRDTARSVHDLAHMSPSDIRTTRRLGSPMARTNRTVALV ELRVPGEDGSAVYASGSGGRLSPVQRGALEELGITIIGGIGHAEQNLSAAIPDGAEVLRW GISWGGTQNPNPCTGLCEPIVNRLGGVIER (SEQ ID NO: 37).

[0304] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38, which is the amino acid sequence of DaDa01 (Accession: tr|A0A2T4Z6L8|A0A2T4Z6L8_9BACL; database: UniProt): MKGREISKPSDTEQKRYNYTLYFLMILLLSIVFILLLPPDIAQAHNCGDFRDCYSTLRAAV AATLGLSMFAVMLSIGLDFVPIVGQTKGMIEAISGRDLITGEELEDHVRLLGILGPAGRIA GGAASVGRGASHFSKAESGYIEIQRFRRILNMPRYSLTNGRTGTVARVEVNGRRIFGVNT SLIKNSKYAPRDMDLRRRWLREVNWVPPKKNKPNHLGHAQSLSHAESHALIRAYERME RLGGQLPKKLTMVVDRPTCNICRGEMPALLKRLGIEELTIYSGGRDAIIIKAIK (SEQ ID NO: 38).

[0305] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39, which is the amino acid sequence of EcDa01 (Accession:Attorney Docket No. GH0197WO WP_001289064.1; database: GeneBank): MSEGPGGPQGATAGGTLAMRMLSQQAMVASQMKRAANDKAIAQMLASKKSGPPAAR LGDEIQHKSFLGALAGAVLGAIVTIAEGCLIMAACATGPYALVLVPALMYASYKASDYV EEKQNQLESWINSFCDTDGAINTGSENVKINGELAARAAVTLPPPPPPGAIPEVPQGEPS WGDIATDLLESAAEKAVPLAKAWGNAVITLTDSNAGFMDRVSAGASLLFPAGPVLMEF ATMVGGRGEIKKEVDFPEAGEDTALCDKENKPPRIAQGSSNVFINNQPAARKGDKLECS AVIVGGSPDVFIGGEQVTYLDIQPEFPPWQRMILGGITIASYLLPPAGLLGKLKNLARLGK LGNLLGKSGKLLGAKLGALLGKTGKSLKSIANKVIRWVTDPVDPVTGAYCDERTDFTL GQTLPLSFTRFHSSVLPLHGLTGVGWSDSWSEYAWVREQGNRVDIISQGATLRFAFDGD SDTTVNPYHAQYILRRRDDYLELFDRDALSSRFFYDAFPGMRLRHPVTDDTSDDRLAHS PNDRMYMLGGMSDTASNRITFERDSQYRITGVSHTDGIRLKLTYHASGYLKAIHRTDNG IQTLATYEQDARGRLTEADARLDYHLFYEYDAADRIIRWSDNDQTWSRFTYDEQGRCV NVTGAEGYYNATLDYGDGCTTVTDGKGTHRYYYDPDGNILREEAPDGSTTTYEWDEF HHLLARHSPAGRVEKFEYNAALGQLSRYTAADGAEWLYRYDERGLLSNITDPAGQTWT QQCDERGLPVSLVSPQGEETRLAYTPQGLLSGIFRQDERRLGIEYDHHNRPETLTDVMG REHHTEYSGHDLPVKMRGPGGQSVRLQWQQHHKLSGIERAGTGAEGFRYDRHGNLLA YTDGNGVVWTMEYGPFDLPVARTDGEGHRWQYRYDKDTLQLTEVINPQGESYRYILD NCGRVTEERDWGGVVWRYRYDADGLCTARVNGLEETILYSRDAAGRLAEVITPEGKT QYAYDKSGRLTGIFSPDGTSQRTGYDERGRVNVTTQGRRAIEYHYPDEHTVIRCILPPED ERDRHPDESLLKTTYRYNAAGELTEVILPGDETLTFSRDEAGREVFRHSNRGFACEQGW NAASQLVTQRAGFFPEETTWGGLLPSLVREYRYDSAGNVSAVTSREDYGRETRREYRL DRNGQVTAVTASGTGLGYGEGDESYGYDSCGYLKAQSAGRHRISEETDQYAGGHRLK QAGNTQYDYDAAGRMVSRTKHRDGYRPETERFRWDSRDQLTGYCSAQGEQWEYRHD ASGRRTEKRCDRKKIRFTYLWDGDSIAEIREYRDDKLYSVRHLVFNGFELISQQFSRVRQ PHPSVAPQWVTRTNHAVSDPTGRPLMLFNSEGKTVWRPGQTSLWGLALSLPADTDYPD PRGELDPEADPGLLYAGQWQDAESGLCYNRFRYYEPESGMYLVSDPLGLQGGEQTYRY VHNPNESIDPLGLAPCPWQIIDRFRSLKAGLSRLGEHIPHQGDGFGTVAYIEINGRKIFGV NSSVLSDKAKDLGRYWVDRLKLGRGVSQVAYHAEAHSLMRAFEKFRGKLPNEITMYV DRYTCPNCQKYLPLLMKEMGIDKLIIYSKNGKVVLLPK (SEQ ID NO: 39).

[0306] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40, which is the amino acid sequence of EcDa02 (Accession:Attorney Docket No. GH0197WO ABG02915.1; database: GeneBank): NTSSGGSVGVGVGVGSGGWGISVSASANQGKGSEKGNGTTHTETTVDAGKQLAIISGR DTTLTGAQAGGETVKVDAGRHLTLTSEQDSDRYDSKQQNASAGGSFTFGSMSGSASVN LSRDKMHSNYDSVQEQTGIFAGRGGFDVTTGQHTQLNGAVIASTATADKNRLDTGTLG FSDIENRADYKVEHQSVGISTGGSIGGQFAGNMANNLLVGANHEGHADSTTQSAVSAG NITIRDTKSQKQDVADLNRDAAHANQTLSPIFDREKEHQRLQQAQLIGEIGNQVADIART EGQIAGEKAKRDPAALNQARAELEAAGKPFTEQDVAQRAYNNGMAASGFGTGGKYRQ AIQAATAAVQGLGGGNLSAALAGGAAPYIAEIIKQTSPDGAGRVAAHAVVNAALSLAQ GKNALAGAAGAATGEVVGMIATQMYGKPVSELSETEKQTVSTLATVAAGLAGGLVGD SGASAVAGAQSGKTTVENNYLIVFEKTELEIAKQTLKNSKNPAEREAAQKKYNALREK DIASDKEVIDACANGNAGSSACAGARLKVFASKEGYEDGPYNSKYNQQYADAYGQIVN LLDITSVDAQNQQQVKDAMINYFMVTKGVDRQTAESYTETKQGLEIIAASVTPILGSVA AKQLSKIVDANLKVVARGNVDGARFSDTNQGVRPSQLADFNKPTLINDVVQAKIDKRP DKNYPNGNMGTAHAEVGVIQQAFDKGMTQGREMAMSVGGKEVCNYCLSDVRIMAEK AGLKSLTIYEEATGNVLFWQQGMKKIENRGPAK (SEQ ID NO: 40).

[0307] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41, which is the amino acid sequence of NgDa02 (Accession: WP_003703542.1; database: GeneBank): MKPLRRLIKLLAACAVAAAALIQPALAADLAQDPFITDNTQRQHYEPGGKYHLFGDPRG SVSDRTGKINVIQDYTHQMGNLLIQQAAIQGNLGYTVRFSGHGHEEHAPFDNHAADSAS EEKGNVDDGFTVYRLNWEGHEHHPADAYDGPKGGNYPKPTGARDEYTYHVNGTARSI KLNPTDTRSIRQRISDNYNNLGSNFSDRADEANRKMFEHNAKLDRWGNSMEFINGVAA GALNPFISAGEALGIGDILYGTRYAIDKAAMRNIAPLPAEGKFAAIGGLGSVAGFEKNTR EAVDRWIQENPNAAETVEAVFNVAAAAKVAKLAKAAKPGKAAVSGDFSISYKNFSTV KPKVIAKGTINGKTFRDVNQSAKIGSPDSPTLIAQRVNAKIQADGKPRPNATVANSHAEI GVIQQAYNAGETKGASMTMTVSGKDVCGYCKGDIAAAAQASGLKSLTVNATDNVTGK NKTYYWTPGMKSIKERK (SEQ ID NO: 41).

[0308] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42, which is the amino acid sequence of PaDa01 (Accession: WP_006660219.1; database: GeneBank):Attorney Docket No. GH0197WO MNKLFYRLIFNAARQMLMVVSDITRSHRASPAGSGENRVENVAKSRVHWSVKLIVTSL WLTLGMVSFSASSSTIVADGNALGNQQPTVVNTQNGLPQVNIQAPNREGVSRNQYSQF DVDKNGAILNNSRTNTNTQLGGMIQGNYWLAKGEAKIILNEVNSRDPSQLNGFIEVAGK KADVIIANPAGITCNGCGFINADKALLSAGKTLIENGKIKGFEVDKGSINIVGQGYNGNG TNYTALIARSVNINAKLHAKDLAITTGKNTVAADGKTILKIDSTSTDDKSEFALDVAVLG GMYANTIKMRGTENGVGVRNAGHIGAEAGDITLSAEGKIGNAGVITASQHIALDSQQSI NNTGTVLAQNDILLNAKQTIINADKGQIVAGRDAVLRAEKINSDRTALLAAGVDSKGKL TNTGSLAVKGDKTVVLQGDILAKDELMATGSDLDLSHSNIQAKNMKLAASTADIRTQE AHLRATNNATLIAKRGIDNQKGEIVANQLTLSAPEFIDNQHGKLVQIENSKNTLNTKAFN NQQGEINLAGDTSITTQQLNNQSGQLFIREGKVDIQSQNLDNRQGTILAPGKQGLILKAD TLDGQKGEILTRGTLGLTAKSVNLDQGTTQAEQITLQANSLSLRQGNMLQTGDKAMTL NVVNGLDNTQGSVASKGDLNVQAGTVDNTDGKLLTSKNHRLDLMSTGELNNTRGVVQ TDKTLMINVDKLINQQGKISSLSGASLLTANHLAGEKGTIFAQTALRIESADINLNQGFTQ ANQVSIFANNFSHQGATLLQTGEGKTELHIQNQFDNQKGEISSNGQIDIVANGLNNQDG KIIAAKLGHLTLTIQQVLNNTQGTLLGNQGIQLAAERLINQSGKILASFGDNQLALKQLD GEKGEILSKGKLALTGEELNLNDAVTQADNIQIEGQNLSHQRGKMLQTGSEKGQIKLAH TLDNQSGNISSQGTLRVNVDKLSNQQGVMVAAKVGALILNAKQHVDNTQGTLFAQQD FTLNTQSFTNIDGKVISKQGDIRLATEDLQGQRGEIIAQGDLSLSGKDIDLTAANTQAQHI QLTANRLTHQNGTMTQLSERQGTIEVSQQINNNAGDISGNGSWLIKTHALDNQQGKIFS ARMGKLDLLIQQTLDNTGGTLTARQGVFVETQSLINRTGKVIASMGDVTFNSQLLESDD GEILAANTLNIEGRTLSLNQAVTQADNIVITANSLEHQGGKLLQTGDKAGKITVQGALN NQVGEIGSNGDLSLTANRLDNRDGQIITAKKSHLKVDLIHELLNQSGALIGEYGLNITAS DIQNQQGKLVARHGDAKLDVAKGINNQAGLIAAEQLLQMSNQALKNQLGYIQANTIDI NTNNQLLDNTQGSLLAKQQLTLNSGKLENQKGTIQSGSDMLIDTHGQQLNNTQSGDNK GIYSQGGLTLTTGGLNNEQGRLVAKNTLALNNQELNNQQGLIGSQSANKMQTQQLDNS QGTIKGQSVNLDTQGQRLTNLANKAEHGIFASQNLAMTVGELINRHGHIQANQITLNTQ QNKIDNFQGEVLASETLQANSGEIDNQQGRMQASQQIAINTNGQLLNNNNTQQSDGILS GGTLTLTTGKLDNQQGQIQSAGAQILNSQLLDNQKGSVYSGSSLSIDTQKNSVLNQEGV LVSQAQLTLSARNLNNLQGTVQGKQGVNVHAETIDNQKGILLSQASAEVTGSSLNNRE GTLQSQGKTNLTIAQHIENQQGKIQSESELVISGQNLDNTNGTVQGLKNIALTVVQQLQN QQGWIKANETVDITALSIDNQNTSQTAKGIEGQNIALHTTYLNNQTGIVRASQTVETTIA QKLNNEQGMISSENQITVTDGTNGKVLAISNQQGTIVSNGSANISANQLTGNGKLIAQKTAttorney Docket No. GH0197WO LTLNLKQTFENVGRLQAGEYLTANFAQGVTNRGLISSLGELVLTTSTIINQVPGEISGQST RIQASGQISNTGLIDGSLTHLVANSLDNRGTGRIYGDFLAIHGNDLANDKQNDKAAVIAG RKHVNLAVENLLNRDHALIYSDGDLVIGNTLDDQLQVTGHAKSVKNYSANIEAVGNLT LKTALLENKDIHLQLTDDAMEVSREHFDWYDFGNGRRYKIQPRSGNQTRYAINDDGTL NRNVGIHYEESNRWRMFVHGDWTKDFYEYVYDRIIYETQVIKRDAALITSGQHLTIDGQ QLNNENSRVVAGKNLILTGYDLNNAEAQGVLRIVENGNTIYRYKGGGTWETYTSTSKY QGVNSEEDLALHLLEVTENAGGINKTQLDSVKVNQLDGQAESVSDANSQENQGVNVTE QALKEGKNTPLDLKPGQQMEVTQLPSISAKVDVQDDKSVDSSLHVDDTNIGKEADIQG QVDAGQGKKITVEAEPQGKDNLDTVIRTVGPNTQLPDNSLFNLTPSSDSQFLIETDPRFT NYKKWLSSIDIVTHEQLHKRLGDGYYEQRLVRNQLIETTGQRLLGNDKNDEEQYRALL TNGVAFGHQFNLTPGIALSPEKMANLTTDIVWMVNKEVTLPDGRIEMVSVPQVYVRAR QGDLNGNGALLAGRNISANMTGKFLNSGEISSRELTDLHAENIENSGRIQGKDVQLDAL KDVKNIGGEIRGLDNVSLSVGRDILSETAQRGDGQSQWLDRPASIYVTGDNGQLTLKAV HDISFIATDVGNLGVEGKTTIIAGRDISLETRDVSSAFDYTHNSSNYYRGANSTEVGIQIQ TQGDLTLSAGQDLSARAANVTSDGELAINVGRDINVTSGIESSDYAKHTKNTEKGFLSST TTETHDEGHERIAISSILSGDRVNINAGRDVNVQGSNVLGTNGVSIDAGNQLTVSTSDET LYATHMNKTTKSGLMGTGGLGFTVGTHSQKVTTESDSNQKKGSVIGSTAGDVTLTAGG SANIHGSDVIAAKDIDMTGSDVTITAAENSRTDITTVESKSSGLTVSLGGAAGSMLDGMA QTAKSAKQEDDSQLAVLKGMKAGLQGVQAQQAGELAGLKEGGSTADAFGVNVSYGS SSSKSTTKTEQHTASGSSLSAGDNINLTATGKKDGSQGNLTVQCSQLDADKNVTLTAKN DINLTSATNTQTVDGKNESKGGSIGAGISTGGWNVNASVNKGSGFEKGDSQFYTDTKVT AGKQLTLNSGKDTTLIGAHASGETVKANVGGDLTLSSQQVTDKYDSKQQSGSVSGSIGS GLNTSASVSANKTEMHSDYQSVDKQTGINAGKGGFDITVGKHTQLDGAVISSTADANK NTLDTDILGFWDIKNKADYQVDSQSGGFSTGGASVGDQFVTNAAGSLLTNVNNKGKDS NTTHSAVSEGEIIIRDKANQNQDINNLSRDTDNAHEKLNTIFDKEQEQKRIEKTQLVGEL GKQITDIAVTNATINATKEVNANHPTLTGKAREEAIQAEINQSGWGVGGDNRRIVEAGT ALVQGLANGDVSKAVANASAPYIANTIAQHIGEENKAGRIAAHGIANVALALAKGENS GAQSLGAMTAEAVGMLSTELYGKDVSQLSEDEKATVSAFATLAAGIAGGLVGGDTSSA VNAAEAGKTTVENNFLSATKNEKLMKALDDQKEGKNLLQASQNIVYLTNEDRASNVL LEQYRNDQLNESQQQDLANQLNQYGYELQTVYGFSETEAKAAIQNLVNGGAFVAAYA DAKAYNEALSYLKMYGVQSGQAAVGADALVALPGVPGSIIRNTLIAGGSYQTGTGVGQ VIEGNYTEGGINIGLGSLAVFGGIAGNKVVEKPTGSIVSPETTIVQESSKILDKKTHTSIPKAttorney Docket No. GH0197WO VEAELIDKETGKIFKDTNQGNRPDYFLGDKSRPTLINDRIEAKVEKNPSKYLPNGNMASA HAEVGTIQQAFEDGITVGRDMNMKVTKEAVCGYCRGDIAAMADKAGLKSLTVYEEST GKTLYWNPGMKSLKEKK (SEQ ID NO: 42).

[0309] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43, which is the amino acid sequence of AsDa01 (Accession: WP_005802165.1; database: GeneBank): MNKNSYRIIYSKARQMFVAVAENVRSQTKASGQSEASAQSNIDNTESQAFHQLWQVKA LVASISLWMPLAPVYAGIVADSAANAANRAVIGAGKNSAGTVVPVVNIQTPKNGISHNI YKQFDVLAEGAVLNNSRQGATTKTVGNVAANPFLATGEARVILNEVNSSAASRFEGNL EVAGQMADVIIANPSGINIKGGGFINANKAIFTTGKPQLNADGSIKQFTIDQGKITVSANP NSKFGLGGNNNDANYVDLYARALELSAELRAKNDIQVIAGANNVSADLQDVTAKTGT GTAPTLAVDVKALGGMYANNIYLMGTEKGLGVTNAGTIQAVNNLVITSAGKIEHSGTIS STSKTQGLVNIQTTGTGAAADINSSGSINSNSMLNIDSGNNLNVNAKEIIINVGSLASSPLI VNAKGNINLAADTRIMDDSQGGDVYIDAANINLAAGSELKSNRGTATIQVQKDLVAAK GAKLIAAKDLNILSNGKLSLTENHIQASLGSINLQANSANTQNLIDLQGGTIYAGKDLNL YSSGDLNLKNLGFSLENAATRVKNIKAYSGRDLVWNNADKALPLITGLVQLDAANNLT VTAKEISNKDSIQLHANQIALNSALTSQKNIDVSSEIADLVLSQALKAQGDINLTALAGG VTANSLKATSSAGKISILANKNINLNSTQTSKAMPSADKDELTTDQTVISGLKGVTLGSIG DGTVNLQSVQVNANQGDILVSSNNGINLKANNDVVVTANKGQVKTVNNVLKGQTVSI ENSKSDIKVQNTDLGSTVGKLAVNSRAGMSTIIDSVLTSKGNTELYAKDLLTLQGVNAT SDQHLAVSSGRTVYSNAEYTPATKWIADKVTNLTSKGVTSVTATGNQVLQNTNLTGGA VLLEAGGFILGQTGLNLNAVGSDLLKNHTKLNSLNGDLTIQTNSNLTIDPKVYSLKAVG DIELVSKTGTLTLKGYGGTAGNGSEQVVKLDTANGGINLEGAKVELQGSNLKAAKDISI VSTNGDLVVDGVRNTITNQHTDEYKAKIIKLDEEIKILQKSSDYEAIIAFFMKEALKVYA RISSGELIEGSPEMDRELQKALQKTTKEFPEYVSKYGPLRDQKDFYSEMIKLFNVGYVNG EVHSGSKIISNTGNIQLVSNKGLSISGSSMDAKDGKVNLEAAGALDGEGYNIQGKYNNP TENSVKKGVIKGSIIIDANQDSYEQGSPSDNDYRWFSPVSPTTINGTKGVSIKVIGKQSTD NLILQGVAITSENDVNIEAYKNIIFDVAINNSFNKSQKTETKSKWYGKKKTITTTQTDEKS GGLSVDIDAKNINIKSQAKNTPAMSGQNRTSIDMYSSQLTAIDGKINIQAGGDLNFLTAD DVSLNTTDISKKSSFLGVKLNKSHTTNTRNVKSELPAELNADYIGTKSGFDTRLKGTVFN YLEGAEIQAGGTITLESASTTVTETLKKESNSVVWQSMQDKGSITETAQLPSFNGPVLPTAttorney Docket No. GH0197WO FIAAGGLSVQVPISEKDVNKVELRNEILKLANQPGNAYLKELVNRKDVDWQTVLLTQK DWDYKSQGLTGAGAAIIVIIVTIVTMGSGTAAAAGAAGGAAASGTTVGLGASMLGAAG VTTTAAGAIVPSALGAMANAAITSLAAQASVGLINNGGDIGKTLKDLGSKDSIKNLAAS VVTAGLMSQVGSALNLKPDSTLLPDRLANNFASSVGSTLVQTAIKGGDLGDNLQVALL AGLAGALQGELAQNLKGLEDVNYILHKIAHAAVGCAAAAATKQSCEAGAIGAGVGEII AQEMLHGRNPTFVSAEEKAKIEAYSKLIAGTISAYAGYDVNTAANSATIAIQNNAFKDK ALLKLEATRKYLDDKSKAALDGLINAYKKGDIEAAKKFKNQLDDSVTTWANSSSSYLNI ETKSALGEIVIVVSDLVIPTNYVEILPVGKLSKVAKILKIGEDGTKSAGRLAEELAELQKV DIKFGKTLPGAKAPITVTAESNIGGKHMFDTNQTARPEVNRTNTPTLAAGNAKIDPSNPN LTMKNAHAEIALIQRAYDAGLTKGETMQVLVRGKEVCDHCGQVMKTMYERSGLAKLII HDTTSGSTTTYYKVIDAKTNIATTKIVVSPIFGH (SEQ ID NO: 43).

[0310] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44, which is the amino acid sequence of HgmDa01 (Accession: JAEZYO010000802.1; database: GeneBank): MSAYEPLLKKPKAPPAPLRATVASKRPQPPIQTALEVSQPGDALELEADRVADHVLRSA APAPRMNAGSDARIDREADKEEQDTEEEYAPNHNSLVVQAMLQRMCAKCDAEEDREE ETPPGKLQRQALVVQRECGDCSEEEEEEQVQREAATPGPTAAAATPRFQAALSSARASG GEALPSAVRGFMEPRFGQDFRGVRVHRDAAASELTERVHAKAFTVGKDVFFRRGHFEP NSEQGKRLIAHELTHVVQQREGMRSVQRELLQREELEAPPAQSAQAAPAEQAATEEEAL LANDEMYLEAHARGFKMAVNGTTYKSWKDPALEAFVLYFLRRYIGGSRDAALACLGD LRATGGIGFQSQLKPGQILNDKGIYGIEIDGALVQSSDAWVKQQRPGLRPVKPKLGIQKIP DIVSEGGGAADSAEEAVERFSPEGRILFPLGDGKIPTFVGGAEIKVAFQFTGAGAALGFN DFPHRINRVDFDWSISTETEGGKKEVDSGPLLEGGANQYSFDVEPGGYLVSVHLKSEHF QSGTELELKRPFEVIDEQARLGEAFDERFVGEGEAQPFELDQEGNLRLKPGAPEARSLKD QILELEFQIGQLEASRDGGGLPADEAAGYIEVFRERIEALKKLEQGSTSSSQYRIHGVFQS REDSSVREIVAVLHGLETSKEGQSSYAFTLNDFTLSPGKAVQHVGAAPAPAGDDRNAAI ASAEGAALEALKERWRANNDYPDGHVRFVVPLKHGSGVKELTFDTHTLRKDARTALT VAAIGAGAVSIVLSGGTTAPVVLLVLEGVSLAAGAALAADEIARAIETNTLTKKQLVLN MLTLVPIAGSLARAGGITVRGMQLIQVSAGVAEGVLITAETQESLRAVMAEFAVQIAKA KTPEQRAQIERQRDQVIARLLGAAAVSGGISLVSTALGARGAIDDVRGPKPRAAVEGAE PHVRAGEVEGPTARPVEAEPAAPRRPAPDVETPEARRRGPVADPANGKAATAEATVLTAttorney Docket No. GH0197WO ERHLTLAGETHTLSVVRKSDGRHLLTLCSPKPCGELANEIEDLLAQLATDHPARRELGRL HEAARKAQADLDTGQLALDAASIEANRIATALDTLSTKHPEVGKILATGGGRAETRQPA EASREPQAESLEPVGKTEGSGGSDRESLTPEGAWREIDRFRTKAKGFEALGDRVPQRGD GLGTVALVEGGGTRHVFGVNSSSLSESSKDLGRKVFSDMKSKGFFPDTKHYGQGAAQV LTHAETHALMRAWENSGRRLGKSVTLFVDRFTCANCRIALPDVMRYMGIEELTIVTKSG VVIPLRVGG (SEQ ID NO: 44).

[0311] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 45, which is the amino acid sequence of MsDa02 (Accession: tr|A0A2G2M3J8|A0A2G2M3J8_9GAMM; database: UniProt): MNNRRSHKQVAGTPHWTARLAVYGLSFLLASQPLMVSAQTIVAGTTVTTQTTAANGVP VINIATPSAKGVSHNQFQQYNIDQQGLVLNNAKAGTITQLGGAINANPHLRNSNAASLII NEVIAPNRSQLNGYSEVAGAAANVVIANPYGISCNGCGFINTPRATLTTGTINLHNGELT GFTVNGGQINIEGQGLNADNISQFDIISRAVTVNADIHAQKLTIVAGRNQIDYATLNATK LNSAASTTVDFALDSSVLGGMYANSIRLIGTEAGVGVRTLGNLATDSGDLTLSADGKIS VNKSQSAHNLTLISANNEIELNDRVHSNNVIELQSQTTVSNNAAVTAGQNINVTATNLE QQGTIASGVTVQGQNYQITQVGDLTLNISNTITNKGKLLSGGYTNITTNTLNNQNTALIH SGQTLNINADQLHNQKDISSVDKLTVQLSDTLTNSGSIESSANLAIDVAILTNNQGTISAV GTADSHITATAALNNNQGTIAANAGTLSLSGQTISNDSGQLLHSGDGALRLISSGIISNRN GNLASNNTLAIVSGTNKDQAFDNTTGIVAANEIKIESGDVSNQGGTIEGNTLELMTNGDL HNGSDAQLNTGFISALSTNDDSLKLNVQGQFNNSTGTTQTNAQSATITANSLSNNGNLIH AGNGTFNINSTNTIDNTAGTILSNGTLNLAASELNNTAGEVRTDGGDLNLTLAGDLSNTS GLIEAGEALAISADNLSNVDGHIKSIGTQDASISTTGQLDNNLGFIESNADNLTLSGNNFS NVNGHITHGGTGALLLNVGASIDNSQGNIASAGDFSQVGFTQFINTNGALINARTVSLVG DTIDNSNSEIVAGDIYIGVETLNNTDGIISSQNYLSIGGITNLINTGGWLQSSDTLDLTVYA DFTHEGNINALNYVGITADNIFIDTTSLEVDGDLNLTAWGDITNYASISSNQRLSINGGNL DNNGSINADLSVDLSLSNIVNNFEIGSYQNINIWAQLENYGGIAAGNDLEISGDIDNYGAL FSGNDSRLYAYNLYNHEGANIFSWNNLTIADYDYYGSNTIIKSNSLINHAGTIEAFNGDIL IKTRTLENKTDLPTITSTRQVTNSNLIYHPQIGGAIYTGSVGMTQAQGYPVLQASQPDAL AIFETYGSNAAWNSFWTRGGSKRVKWFDVNVTRTRQQADLNITNPANLVAGNNLIIEA DDITNQLSLIGAEGDIHMTGNNLSNVAFSLTDTITGNWRLGREKKRRAVPRSEPSQNNY YEQAGSINTSTVIGTINSTIQAGGSLTGDFTHRIDNQNIKQNTAGLTHTEQFGMGGGGFGAttorney Docket No. GH0197WO TGLTAITPQGVATISGSTTPAVLNGNLAALTPVSLQTPTFTLPTGSGLFVVNTQPNHPYLI ETNPTFLSNTNFTNNLTSTNHLFTGLGLQADQFTQRLGDGFYESRLLRDAIFAQTGRRYL TGQSNDQAQFKYLMDNAISARDSLNLVLGVALNAEQVARLTHDIVWLVEQNVQGHSV LVPRFYAATLNANDLNGQGALLVASTVELTGQGITNTGTIESDTNLQLTSNSDLLNRGTL KSGTNIELVARNNLFNTGGSIFAKGDIVLVASQGNIDIGRYTEQLTSTIDGRLQLTDTSTW VGDASTIQAGGTIDLHAGSDINIAGSTINADKSINANAGNNLNIQTVATTTDVHGQSANG RFSVDFKATTLDQLASQIQAGEDINLTAEEHIAIIGSTVTAAGDTNLNAGSDIQIAAAQQT SSYNYDSKSGGRTTKIEDRSTTHQQATIDAENININSGRDIAITGSDVNASNDINLQAHDN ITLASVENSEYHYYFHKAKKDFGRKKSETLETEQIRNLASSLNAGNDVLINVTKTEEDAE TNTVLSLNTQANDVHIIASDINAGNDIVIYAGDNLSAQTSTDYDYRFHEKTKTGLFGLTG SSESTTTKATRQQQANVKSEHDISLNAGNNIALISPTLDANNNIDIQADNQILLGTSKDSD YYHYEYSKTGAFKWEFGNEGHYDETVRHTEITAGGNINIDAGNGTIIEYKQNGTLEQSIN TLSQLPELAWMKDIQTQHNDTVQWQAIIEEHDKWQEHESGIGGPGVTLIAIAVAIAMPT FGADFLGYTGELTAIEAAQVAVIESLANQATVSLITNGGDISAVFEDLLSKEGLLNLATA GATAGITNSLYTKLGLSTEAINLNDSLVANGAGLTLAENLQRNIIRAAVQSSVNTVAYG GDFEKNLFKAAANATVNTLAVQLTKDIGNLELTDGNISEGGLTKILAHAAVGCAAQSLT GGNCSAGALGAATAELLAPHLSNSSLNNVSSELSVLLATGIAALVTGEDLEQTINAGLQ VERFNRQLHAGEIALAKKNAKLLQAAAAKRGEVISISEAEARIEKQLLRWVDADSNDGK VDELVISIIGFKGGNSDFDWDYKNYASLNPEQYTDSSINSENIPYYASELPVSEFGLTPEQI KERNDTLAAKVAKIGAFVVGVGSGAEIGIVLVRSASTLAAAAKVSIVTCLANIYLCGTQ ASIATGEIALGDAIGAASVLGTGAVATEKVGSKLIDELVEAGRKLKNDNAFDIIDTFRNH KQGMSELIDHIPVQGDKVGTVAVIDVNGQRTFGVNSTALVNDVDKNLARSWRERLGLN KGDAQALFHAEAHSLMRAYQKSDGKLPANVTMYSDRQTCSFCQKNIPALMKEMNIDK LTIFFKNGASMILRANN (SEQ ID NO: 45).

[0312] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 46, which is the amino acid sequence of XinDa01 (Accession: tr|A0A1N6MQY7|A0A1N6MQY7_9GAMM; database: UniProt): MGLIDDIVTRSARVGAIHASQRGQGPKGPAAAREGDPIQHSSFWAALAGAVTGALISAA IFGAAALLVAGTGGAAAVVLTAALGTAGTMALGDLILGASSAVTDMINSFCSPDGMLTS GSPNVLVEGKPAARATVDTAVCSKHPSPVPPLIAQGSETVFINGQPAARVDDKLVCGAA IKGGAGTVFIGSGQHAYLDVQEEFSLLERALLVAVEYLVPPTRGGLKLAGKLLTQQGRKAttorney Docket No. GH0197WO VVTRAALKEAKAMARDAKSILAGAKQGAKKAQQWAKNAAKNPKALTQKAMAGAKA GAKKAAGNVKAGTQKVLKGAKTGATNAGKKIKDNYNDRVGCAKKAFHETKGAKRY WEATKRFFKGDPIDVTTGALFDQRTDVELGQTIALTFLRSWSPGESGLLGENWTDSFSG CAIVIGDRVEIRTTEGASLYFILMATDLNSVNPEHPQFTLSRANTGYVLHEKNNPVRQHF SVEVSDNKECQTWKLSSLSDRNGNAIDFHYSEQNQLIKVTHSDGPELVLLYRQDGLLKE IRRSDNDLNEVMARYDYHDNGWLAEADSTQQFHLFYEYNEQGLISHWSDGDQTAVDY IYDEQGRCTDSVGSGGYYPVHFEYEPGITRSMTPQGYTTIWHYNEEQLVTQVETPCGHIT HYEYDEWGNLRRQILPEGQIITLDYLADTGLVTAFTDATGATWQYRYDDLDRLSSMTD PLGRTWKQQYDDNGNAECFIAPDGSKTTLIRNEFGLVTAAEDDEGHKRTWEYDEHKRL TKLFDEENRSLKLGYDSHDRMQRLTSGSGALWLWEYDRHHRVSLSDRPNNSIERFRHD RHGNLTDWTDARGVKWHIEYGPFDLPMARIDGEGNRWQYRYDLDSLQLLEVVNPQGE SYSYILDADGRVITETDYAGTQWHYAYDGNGNCIEKRDALGNITRYEYDAAQRMTAM HTPEGTTTYRYDILGRLLEVIAPESAPLTFEYDDKDRIIKETQTHGKIERDYPDNATVERT LRNIDGQCWQVKAEANKVGELRLLSINGEHTLDLERDEDGHEWHRQSDKGFILRQEHS LMGQLTAQRAGRNTEFFASHEVADIPQPTLAGLDREYRYDAALNLVAANDERQWLRY VVNGNGQVTSVSEGERLREHYQYDASGYPARRFDGVNEVDGERLYQKGHRLRQLGQH LFEYDDAGRMTAMQLWQDGHRPQLTKFRWNSQNQLIGVQTPGGQQWDYRYDAFGRR TEKVCERAGSRTTYLWDGDVPAEIREYRHNRLYSIRHLVFDGWQLLAQQVQFFSLNPEN RQELIAGEIQTQYAVCAPTGEPLALFDAAGHRVWRQPPHSLYGLRLGRLGENAELNPGL QFAGQWLDDESGLVYNRFRYYSPVASCYLTPDPIGLAGGEKPYAYVGNPAQAVDPLGL AGCEDPWKIVDRFRRSKNKMEPLGDRIPGAIDKDGLHTVAFFEMNGRRVFGVNSGTLY KKDKALGKQWNEKIDYLTKEEKGTSAFHAEGHALMRAHKKFGGVMPKEITMYVDRVT CNHCERFLPALMKEMGIEKLKLFSKNGTSSVLHAAR (SEQ ID NO: 46).

[0313] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47, which is the amino acid sequence of XjaDa01 (Accession: tr|A0A1I5CFX1|A0A1I5CFX1_9GAMM; database: UniProt): MTALQLWQAGHRPQLTRFRWNSQNQLIGVQTPGGQQWEYRYDAFGRRTEKACERAGL RTTYLWDGDVPAEIREYQHNRLHCIRHLVFDGWQLLAQQVQFFSLNPENRHELLAGEV QMQYAVCAPTGEPLALFDAAGHRVWRQPPQSLYGLRLGVLGENAELNPGLQFAGQWL DEESGLVYNRFRYYSPVAGQYLTPDPIGLMGGENPYAYVHNPSTWVDPMGLAGCPWE QIDRFRSRTRLPVLGNRVPAPNDGLQTVAFVSIGNRKFFGMNRQAIPGLEYAAKRKEWFAttorney Docket No. GH0197WO RKIYKSTGVEPSTISKAAADAEGLALVRAYERITKSGGKMPKEVTMFVDRPTCDFCKNY LPLLRESMGIEKLVVYNKGVSNPLIF (SEQ ID NO: 47).

[0314] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48, which is the amino acid sequence of RhDa01 (Accession: tr|A0A1V4SQ45|A0A1V4SQ45_RUMHU; database: UniProt): MKPLEGPVNSVGGMIEQVTDQATGVVDNAEQSAMSSMGNGSKLLSGFAGSSGSQNRD AGKAGESKGQEKNSGKSGQEGGKSQGGGDFLGLLKSGVHTRLMTFGLMNIKKLGAKVI AAGAAKVTGVIKKMLTPKVKFRLGAEEHELWVEKGKNGNVVMMASKKPGPIKRKIEE GEIPDNGEISNKRRKVEAEKDEQQVVQQNEAMASTIQAVTAGVGEAGKKEPWEAIDRF RKSNGLEPLGDRIPVRGDGLETVALMEVSGNKVFGVNSSLLSDELKNLGRDFFKVIKEK GLLGNAKHYGSGEAQVLTHAEAHALMKARKEAGGHLGDSVVLYVDRLTCPNCQKYLP EVRAAMGIKTLKVITKGGIELIL (SEQ ID NO: 48).

[0315] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 49, which is the amino acid sequence of MGYPDa04 (Accession: MGYP001129217467; database: MGnify): TFTGAVPTNGSLSLGASEANRAWADVLSGLFASGSNDIFVGGETALIGAVLAAADGDLN LDTGSLRTEDLFDTDTSNSWGLSVAGGTGFGDGSASLSSIGGHYSTATREAETNATITSG NVTVRDLSEDETTELLASINRDVSNTQVITRDSDFGTGQVNLEVQSILEIDDNLRAINDLR RIIAPPTSVDSDIKATFYRRLIANGIDADQAEKALKSQQFEEFSTVSQNWDRAIKAFGSAG DVPKNVRALIAMGKDVKLLFDEDGNVFVENENCAPTGDSGCRLRFGGNLSDQQVEEIA STALKNIKKLVEDLESIKTRMSETGKKLSELQYLCATYCDPRGDDRILEIEGLESDLNEFR IALLVGEIAVNNNLRYYALCSDPSESSTSAFAASISAALGKELGDDHGIGSNQIQEAANFG IAGDRAGQRADDSGQMLEMDFANAVFQRQDPLDELLNPSAAGSVIDAGIALRAAMYG GSRGGQYTEDLTSRSGARQVDPDNIVRAMVGYNIALNNLVDEVNGEDNLDRSTLFFSD FDDASHVGLSQNEDGTVGTDGNFFTRSSGDVGGEFRTNLLLAFDQNIRSAATGDTQLNG TIEGATGLFSLNNGENWNARTRENSYLQTLVEVLQAEVAGYGAFAALGYLGTGARTAL ALDNVGTITRLDGPATEITARAAGRTSPNPSSYTDEEAFDLINRFRDKKGGMDDLGDYIP IQADNTGTVSFVDLNGNPVFGVNSSALDNTSSAYQGLMDEARDWRKILIDEYPEDMAT RNVGSKPNDSLFHGESMALIRAHRKSGGNLPTEIDMFVDRATCNSCKTVLPLLAQQMG VQIRLTMKNGFKGVIKDGEFVPSPG (SEQ ID NO: 49).Attorney Docket No. GH0197WO

[0316] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 50, which is the amino acid sequence of MGYPDa05 (Accession: MGYP001094202578; database: MGnify): KSGFGSADEAFDAIAEFRSRTTITDTQGGRGTVAVTEINGQRYFGVNSSNLSGADKNLA RSWRDRLGLTRGQDQVVFHAEAHSLMRAYERTGENLPSNMTLYVDRNSCGTCQTYLP RMAQEMGIDNLNLSFTSGRTAAIRNGAFEWVN (SEQ ID NO: 50).

[0317] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 51, which is the amino acid sequence of BaDa01 (Accession: tr|A0A410KXF4|A0A410KXF4_9BACI; database: UniProt): MDARAKHYQELREQMVDLKKALQGVANLGDDFTGKGADNIKSFYKELSGNVDMFISFI DKQIAFHDGVSGTLDDTSFGGDTFVEEHFLDNAVHMGIKNSKSIVKDQKKALKTIFEDID DLISLEVFDSKTFDEKIADAEDERKKTVKDLIELDQNLKDEYALSETEEKATMALYAEMI NATNDGKSISPMNFDKKAFQDSEIYKAKSDIEKQTSEYLKIKKDQEEAREIAKEQEALAN RPWYEKALDYGGNIVNELTGVNDAKRAATGVDPITGEELTSGQRVAAGGMAAAGYIPI VGWAGRIFKGGKAIYKTSKATSAAVRAVDIYKTSQKSFDALKTSQKGLYGLTATNGFSE AITGRDMFGNKVSKEQQEASMNTALAILLPFGMKGPIKKTPGVIQKSNKQISESYDEIKR FREKAGLKPYAIDSGDTVAAVKVNDRTFFGVNSTITKESQQATRELRQKWLKEVQWVP PKKSSPKHLGHAQSLTHAESHSLIRAFEKEGQLPQKVTMFVDRKTCNICRGEMPALLKR LGVDELEVYSGGLEKPLVIKATK (SEQ ID NO: 51).

[0318] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 52, which is the amino acid sequence of WWTPDa04 (Accession: tig00000754-10-8122160_273; database: GeneBank): VNWRVLTPADQFELHGYFQTNGNTDIDEIAEGFNLTGTGLLGVSVVPTPQTKAIGGIGA GLCFATSSGLNLYQRTSQGFNTRHDLLENGMDLAMIALSLSGAPRIAGNYLAKVHTGKL QVFGEGLESLFLHGKKVVVLAKLEVGAGRSLLCVCGIQAIQSVGSIVGRDDLSTDEKFH LLVETLGLFALQGLLMVVSRRTDKYVKYPTRSAESPPPPKAMGKTDHSPLENGKPLEGE SRSLKHEGEKPPDADGRGKVGEGEKTSDSENGAHPETEKQPEGESRALSEEERSSPNESK PKSMESETNLDEIPGQTGAERHETIVSRGKRGKGEPKEDARKPEEPPKPEPVVEPEPPKA WPRETDAPPPPFEKGKPAERSWNQWFQNERMPNVPNVKLGELKVVAYAEINGKQFVDAttorney Docket No. GH0197WO YNQNARLKPLADEQTAIYEIIKRRASKLGKEISGFPNWKHLNLHAEGGLLYQLYRAGLT RGAKIVITVEGRKVCGFCLKDLIELAKALEIKELTVFEKNSFNELPPETLFWKEGENTWT DLN (SEQ ID NO: 52).

[0319] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53, which is the amino acid sequence of PbDa02 (Accession: A0A356KTR0; database: UniProt): MSHATSPSGLVTRFTYRDPTTDPDPGDPDNRLRGPSLIESREGSGPAVWTIDHQRSSSPV GFVVTVSDSLGRSAIHSTPARAAINKGRYTSSWTTVAGDWSRKVDLFSDRQSVARRIPF NNFSFASTYRFFDEGRTHIRVLGFEEGGSRAHLALDGFWTTQVEPLQGPTQNYAIETKQ GGKVEGLIRNVSWEVAPGSLATVGITRNEDGEVEEVMNPVGVTTTTERYLSANKQGLPS AQIVAPGGTTYLPLGATPLREEFDYDLMGNVTSHRDTAGLATITAYTALDQIREVRLPSG LVQTNVYDQAGRLQSTSTNQGGRVTYVYNEFDQVIRVDQEGVGSTHYQRDEMGHLTQ VTTPRGEEVRYEYDNRWLVSATEHHAAEEVAGPLELQRRITYQRIARGNVLHETRTEGS AGANSYTTSYGYTAVGLVRSVVRPPMTLGESFRPQTTYHRGSNWLVNRVEQDVREDRE DKLASYVRDDADRVIAVRNGTAEATNPHRSQSEVLLDLAGNPNTFLSGDFLYDPSAPVG GTVLSDRPRSTVTRDGLARVKEVRNGSGCVIAELTYDSAGRVVVERRPNSNEQLEVVSR NVYDPLTGRLQSSEGYDYPGAGKAKREYYYDAEGRVKKTFVRDGGGNLITVNEVWYD AIGRVVRRQSRDKDTNPTEQRESRYEFHPQTGDVTRETQVVNGNSYTWVYTYGVGGRL ETTTDPLGKVSRLVYDAEGRLQKRIKKDGVEITYEYDGWDRVKTVTGPTETITYEYDAR NRVISEANALVKREFSYEDDLGRLESVKVTFLQVGLQVPKTFTYRYNAAEHRYGGLTEF VDPNGLETKYSYNRELDVERIEQRQGSAGPFSEVGSFAYDCSQRMTEWRTRGGSWERD YDATGRVLRQHGAQPNHSISYSHDAFGRRKTATYAHLQGLKFAWAYDGIGRMFDEQIS YGDGAPAYRETLTFDEAGNRSSRILNGQTTLYDYNEFNQLVRSRERVRTVANLTLSATT GPPASPGTDVLGPSYAPGLAVDGTIPTTSGANQAFVSGTADVEHLLEVSVTSGAVDLTG LEVHLPTDRNLPDKLHLEYFDGSAWVPLPVQSATGADVVMSGSDQDGLRARGARVFA SVWPPINTSQVRVRQAQGEGPAQPTTHDNALAVNEVRLFTASSEVEVETSYVYDDNGN MKGVTRGAYERTYEYDQNDRLISIDDVGGAVPVNMRMAYCPSGKLTRQEVSGSVEHL LYQGDELYARYDASDALTKRYILGPGLDRKIGVVPYSGGVAENTRYFFPTPNGSVHQVT EWDPLRNGGLGAAVLANTELTSAWGVPILQDRIPELLAGGGPPVVSIFAPDFGYTGRRK LGNSGLMDYRNRIYDPLLGRFLTSDPIGTKAGLNTYAYVNGDPVNLTDPFGLDPYDVHP RPEEWYNQPGLYNKFQALRGEIGALWNRHVNPWGDAGAEVLEETLDEGEKLPGAMRTAttorney Docket No. GH0197WO LGEAMQADPGGTTRIMLSQLPGVARNMVMQPIDQTAEAHYYHLLGDAVGERKAVIKS TTGFMALFGVAQGTRSLALRTTGVLRAIREVCFVAGTLVVTQEGRVPIEDVSVGDLVLS RDPETGEIGFKRVTRLFSGKTTEIAEVRLFAPDLTDDSASVVVLTCTPTHLFRTRGRGWV EAKSLKPGDQLVTETGSLAVVAESPSIRQVEALSRFNLEVEDWHCYFVAPNESSASILVH NDCWDLINYYRRVHPNLPDLPSPDYVPGSQKIIPGSGVADKTLGTVAFVKIGERVFFGV NPTAMSDAMKNLHRLFFDTVLKPGGYSGTRSARTGKALTAGQYGGMANLFHAEATAL LRAWKHNAIPKGGVLRMVVDRHTCRLCMKPLGEIVHALGLKGLEIVQKNGKVVRIKPI APGQ (SEQ ID NO: 53).

[0320] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 54, which is the amino acid sequence of CrDa01 (Accession: tr|A0A1S8L869|A0A1S8L869_9CLOT; database: UniProt): MGAGAEAIYKASKDGFKEAKNALRTVKDEKNGFVDGVADTNAPWKAIDDYRNQRQG LEVLPEDYEFIKGDGKNTVAFNDTCDKRYFGVNSTLRTDAEKGLAKKYFNKLVEEGYF PKGSIYPRGKAQFITHAEGYTLIKAYEENGIDIGKSVTIYVDRPTCGFCQNNLPKLQASM GIDVLTVINKNGDVFRCDLRKYTSMTVSDVNAEKLIKIKSK (SEQ ID NO: 54).

[0321] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 55, which is the amino acid sequence of MGYPDa15 (Accession: MGYP001492404292; database: MGnify): DGRFSSRVEKDPLYTGTSLNSQQIAQHLTEKQMTAVAGSADLANALWFAGRQKKVLD VDVPFTIPTEVLADPRDAVIIDGKLQSRKKYAEELRRDAKKSYSWAIGLTSDEEFGYRGL FDRKKSLDSIFPSMLPEDESLVRRRAQERERTKEQAAVWSENSLELAEFLNNDEAFKKA SGSMIVTGKSGKPMLVPYRQASHSMLSRQINAQEKMSQSYYADQEGTLIGLGAVGVVA APAIVFALPAAAVQAVAVGGVTGAAFEAGTQGLEGAFGYRSEFDWGALRDQTGAGMA LGGVLGKPATYLASRYPRATAAVSGSLGVGGGLYEWQVNGNPVRGATTAGLGLLPAP FLLRNRTPSSELVHVPGQGNQTFTHELTGAKFGTVEELRAYSKWLAAGKMGPPEPATPS STSALSPAKAFDAIDEFRRLGQGMQPLGAVVPVKGDDLGTVALIKYEGQSFFGVNSSILS DESKNLGREVFSLMQQQGMLKGATGYGQGAAQVLTHAEAHSLMRLWKKLGSSMPSE VILYVDRYTCANCKRYLGEVAKALGVKSLSVVTKSGETIPITIP (SEQ ID NO: 55).

[0322] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceAttorney Docket No. GH0197WO identity to SEQ ID NO: 56, which is the amino acid sequence of MGYPDa16 (Accession: MGYP001076701719; database: MGnify): MFGCGHAMRGKAGTASCGLMNFLKYGTAALSVGTGGYDIYQIGKENYQKYVVNGEKF SWGDAALDTLRITLDATSIAGGIKLATETIPYCFVAGTLVETEYGQKPIEEIQAGDKVLSE NPETGEIAYKTVEETYINETDELIHVHVNDETISATPNHPFYVDKLGWTLAKNLRAGDIL VLSNGEFVVVEWIQHEILENPIKVYNFEVQDFHTYFVGKSSVLVHNECTPEEAWKAIDE YRVKKDGLEPLGDSIPKRGDGKGTVAFVEVNGNKIFGINSSLLSKPEKELGKKMYEDMR KAGYFSTVKSYGSGHAQVFTHGESNALFRAYNIYGDNIGKNVTIYCDRGTCGICQTNLG YIREFLGLEKLTVINKNGKKFEF (SEQ ID NO: 56).

[0323] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 57, which is the amino acid sequence of MGYPDa17 (Accession: MGYP001100966096; database: MGnify): ITSQYYLRARFYNPVIGRFLNEDTYYGDGLNLYTYCHNNPVEYVDPSGNWCKKKFKQK VKEYLEKGYSKKDAKVMANYEQIRENYDVNSAEQYLRAVLDYENLSPEAWKSIDQFRE SHGLNTLGNGLPKKGDGTVAFINANGEKIFGINSSLLSEDKKMLGKKYYESMKEAGYFD NVTSYGNGSGQVFTHAEGNSLMNVYDLYGKSIGKDITIFCDRTTCGICKNNLGYFKDYF GIDSLTVLNKNGDIVIINKGTYIKIKS (SEQ ID NO: 57).

[0324] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 58, which is the amino acid sequence of BaDa02 (Accession: tr|A0A6B3W113|A0A6B3W113_9BACI; database: UniProt): TGYSSLPIVGMGTKPLGEIRPQMFSVKSKEGREMGNRKSINGYEGIGKFREKVGLKPYSI DSGDTVASITVNNKTYFGVNSTITKESQKASKALRQRWLREIEWVPPKRTAPKHLGHAQ SLAHAEAHSLIRAFERQGSLPKTVTMYVDRKTCNICRGELPALLKRLGIDELEVFSGGTT KPIIIKATK (SEQ ID NO: 58).

[0325] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59, which is the amino acid sequence of VsDa01 (Accession: tr|A0A1N6M042|A0A1N6M042_9VIBR; database: UniProt): MPAAARLGDNCAGHGCFPATPIMAGSGDVSINGKPAARKGDTVLLHACPCPQFPHGMH GRSISAGSSNVSINGKPAARVGDAIDCGGSVAAGSGNVLIGDTPYKSPTHKCGEGAAMEAttorney Docket No. GH0197WO QSPFLRIQPLAEAPPFDWASLPFVEEVYERSAKDKQKVVEKKSKKIRKGTNYNHKIQSPS DGYNVISSFRKKAGLQPYSSTSGDTVAFIEINGKKFFGVNSQITKTSQVSQKALREKWM KEVEWVPPKKNKPKHLGQVQSLTHGESHSLIRAYERLGNQLPKELSMYVDRKTCNMCR GEMPALLKRLGVDKLNIYSGGSTKPIVLESIKL (SEQ ID NO: 59).

[0326] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60, which is the amino acid sequence of MGYPDa18 (Accession: MGYP000969250293; database: MGnify): MNYMPDKAATEAQAAAKANLYIALQKMFPTASKRAIDEVAKTILEKRAKIARFREIAGL QPYSEDSGDTVAMTEVGGHAIFGVNSSITKESHEATKELRQKWFKMVKWCPPKKKEPK HLGHVQSLTHAESHALIRAYERLGKLPKRIIMYVDRKTCNMCKGEMPALLKHLGVDEL TIFSGDSLEPLVLKAVK (SEQ ID NO: 60).

[0327] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 61, which is the amino acid sequence of MGYPDa19 (Accession: MGYP000654575191; database: MGnify): GVSIGSNPEGKSKPGITVEGAYSNSETEGVARATIGEGEIIVRDGDGDGIKAADLEAEAD AAEAEGDTARAEALRTEADREAAEDKTTTETQLANINRDPDAVVVVTSRKEEGFELYLS DTSLEKAVEGIEVTGKALGEAFRALGEELAASGALTPSELDTAKTVAKAIDEGDLDLRA LVTCSGRRGFNLWDLVVSSAHAATGCVLFDENGREIAELTPREREACVQMLSRLLEEYA RDYLVGERGDTELPASITKTAETLREMASDEVLVAGAQSLGMSVSLIRDVSMRLALGEE KYAEYKEALKPLADAGELSSEAVARTVERFAKEQGLSNQEVKDLKLVAAVTTAAIVTA LGAKRVLINGNRAPYTADVAFATIDEFRKHKGGLIELGDSIPLKGDRKGTVAFVDLDGK PVFGVNSSVLVRDTDKELGRSWADTLGVKYGQGTGQYLTHGEAHSLMRAYEKSRGSM PSTVNMFVDRYSCSFCRDPKALPDLAQRMGIENLNLTFKDGSTAVINSGRFISK (SEQ ID NO: 61).

[0328] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 62, which is the amino acid sequence of HmDa06 (Accession: 3300043543_Ga0453689_0082580_8_1099; database: IMG / M hot metagenome bin): MKLQKGFCFLLRICNGQRWRVQETAGGITTGLYYNRARYLDPTTGRWTTQDPLGFAAG DANLYRYVGNTPTLLVDPSGNIWYPGKYIIQYFREWRRGRELDAQLDEIQRRRHDPLAAAttorney Docket No. GH0197WO VMQAGSGPWSIHNTYAIADPAFDRNWWQGMGDAAVLAAVGANWLAAGTTWVSPGG QQLVWQSGRWWNRTANRAATAEEAAAATQAVAAARGAGAPAARTVNHFDEIARFRQ RAGLPAFRAEDSATGTVARAVVNDGTSRAYHGVNTGLSSNSIPSRMEVIQQLQTRGYFQ NVTNIGQAPMLAHAEAHALMRAVERTGARSITIYSDRTVCRACRTQLHLLAEHLGVTEL RVFEAGNPLPYIIMPRR (SEQ ID NO: 62).

[0329] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63, which is the amino acid sequence of MmgDa02 (Accession: JANFCO010000966.1; database: GeneBank): LTIDSVDGIGGIATSGDVFVSGVGLTVNSGINVGANTVRLQTSAGISQGFLGTITAGAVA VSNSGTAAVSLASSNSFGAFAATSSAAASISVTDSVSGGMTIGSVAAGGTLPAINGITTTN GTITIGSSTGVTVNQPILSGGAAVTIDADNQAINQSVNAGTAIVTLSPYNGNRAINLGAKP GGVLGLTNGDLSQITASVLRISNTASSGLTVSAAITAPLGWNALALLSSGPITETGSLAVA NLRVQSSNSSITLIGANAVSTVAVQNFFANTGPVAVVSTLPLTTGTVDGFSGVSAQSADI LLSGAGISVTQPIIAVNLRLLTSAGVSESGSGTISATGIGVRNTGSGSISLPLSNSFQTFAAT GAGEIVANAGGQPLTVGTITADSTGTFTPLSGITTSNSDVELYTSMFGSVAATQPINVGT GTVRVTGDGGFTESGSGAITAAALGVYINNNTVTLDGANNFATFAAAMGTFGSVVSIND AGHDLTIGTVGADSAGLFASVSGVTVSFFPSMTVSAQSLSVTQPIVVGIGNPVRFVTTNG VTQSGSGTITGNLGIRNSGSGNVSLTLGNSLGTVAIAQTVVAGTVSINDTAAATIGTIAAD AQSLFSATSGITTAGADILFTETSAIAINQPLNAGTGIVRLVTAGGISESGAGGVTASALG VINTFSGSVALPQANNVGVFATSNSVNGGAITFVNGTTALTIDSVSADASGAFAATSGIT AIGSGGVTLSSGALSITQPISDATGTVRLVAAGNVSQSGMGIITAATLGVRHSTAGAVNL NLANVGPTQFAATSSLSGASITFTSAQAVTVATVSSNTQFSTTTGIITSGGGNVILSSGALT ITQTINAGPGTVTINSAGGESELGGSITAATLAITNSGSLNVSLGVGNSVGSLSITNTVGNV SFTNAQSLAVTGISALNTSGTVSVTTTGAGHSLSTSGTVSAGTSSGSATLTSSGILTLGGD VAATTVTLSSVGGTSQSAGVITAAGSLGATNTTSGDIVLTQSNLIPSFHATNAVGNVSFM TGQSLTITGITALSTTGTVSVRTTGAGHTLTANSAISAGTTSGSVSLLSAATLTLTANVTG ATVLLSSVGGMSQTNGAITAATSATATNATSGNIALAQANQAAAFTAANPVGNVTFLD NRTLVVTGITTLSTSGIITVTTTGAGHNLSTSGVISGGANSGSATLMSAGTVTLGANITAA TMAINSVGGSSQSASAIVSGGSLDTTNTGSGDVVLTQTNLVVSFHATNTVGNVSFVNGQ SLTVTGITAQSASGVATVTTTNAGNDLSTTGTITAGTTSGAVTLTSTGTLALGTITAATVT LNSVGGVNQTSGLITASGALSATNASSGDIRLTAINQIASLSATNSVGNVVLRDGRSLTITAttorney Docket No. GH0197WO GITALVNTGTVSVTTTGAGSNLTNSGTISAGVTSGSATLTSAGTLTVSASLTAATVTLSSV GGMSQGGAITAATLTATNATSGDITLGQTNFVALLAATNPVGNVAFSDSQSLTVTGVTA LSTTGTAILTVSGAGHNLATSGAISGGTTSGSVVFTSAGTLTLGANVSAATVASSSVGGT NQTGGVITAATSAGATNTTSGDITLTQANLAAAFSATNLVGNVAFSNGQSLAVTGISAL GTTGTVSVTTIGAGSNLSTTGAISAGTTSGSATLASAGTSTLGASVTAATVTLSSIGGTSQ TAGVLTATTAGTTNTSSGNITLNQANLVNSFSATNLIGNVAFTNGQTLSVTGITALSTTG TVSVTTTGVGSNLSTSGAISAGTTSGSVTLTSAGTLSLGANVTGATVTLSSVGGMTQTAS LVSAASAGATNSGNGNITLSQPNSVASFSAMNTVGNVAFTDGQTLVIAGIIAQSTSGTVT ITTTSAGSNLSTSGAISAGASSGSVTLTSAGTLALGADVTAATVTLSSAGGTSQTSGVITA TTAGATNTTSGNITLNQANLVNSLSATNTVGNVAFTNGQALAVSGITALSTTGTVAVTTI GAGSNLSTGGAINAGLASGSVTLTSAGSLILAASISSATVTLNSATGASQTAGAISAATVG ATNTTSGDITLNQPNLAGSFSATAFVGNVAFTNGQTLVVTGVTTLSTSGNVNLTTVGTG SILSISGLISANATSGSIAIASSDVLILAANVTAATVTLSSATGTNQTAGVITAAGISAANST SGDIALGQTNSTAAFSATNLAGNVSFANGQLLAIGAAGITTSGGNIAIFAPILSINQPIAVG TGSVSLTTTAAGVTNTLAASITAASTTWTATAPSNLSVGGVSRLTDDEAFALIDKFRSKA NGLEPLGGRIPVKGDGLGTVAIVETPGRKFFGVNSSLLSDASKDLGRDFFQVMKSKGLL EGASAYGNGAAQVLTHAEAIALMRAWKFNGGKLGSEVTLFVDRYTCANCRKYLVEV MEAMGIKKLTIVTKSGEQIVL (SEQ ID NO: 63).

[0330] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 64, which is the amino acid sequence of HgmDa02 (Accession: JAEWLH010000049.1; database: GeneBank): NLMTLGLINLKKLGMQILAAGAAKISGAIRKMLTPKVKFKLGSETHELWVEQDKTRNS VMMASTKGTEVEENSKASRILAESGELKDKLEKTKKKQNESQANANLQDLAQGIQAAG QGESKAANNGPWEEIDRFRSKTKGLEPLGDRIPERSDGLGTVAYVELNGTKVFGVNSSV LSDESKDLGRKFLKSMQAKGFLNEVKSYGQGHSQVLTHAEGHALMRVWKKTGGNLGS NVVIYVDRLTCGICQENLPKVKEFMNIKTLQVICKNGTKFTF (SEQ ID NO: 64).

[0331] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 65, which is the amino acid sequence of CgmDa01 (Accession: JAEEGH011632547; database: GeneBank): EDYYRFLPEFKKVPITAREPEKPAEKPTLAPTEQSGYTEKGFTGRVQDKITELRMWDSLDAttorney Docket No. GH0197WO SFRETRGLPTTGTEPTKPGDGKGTTAVIMVNGEPVYGLNSGFRNAEEQQFIRECYEELIG SVFEQKENTHYGKGQSYFLTHAEGYALLQVWKNNGDLSGQDITIYCDRDTCGYCRGGL KELGKTLGMNSITIISKSGEAIYYEFGVGMTRWQPLS (SEQ ID NO: 65).

[0332] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 66, which is the amino acid sequence of FbiDa01 (Accession: JAJXVD010000193.1; database: GeneBank): MIIEGKTGTDEKTIPLFEWSMLPATDPNCYKEIVVEQTHEDVLLRRIKFSKAFVVDYSES HHDNVGYFSLHVRQFAGLDIECSDGQPQAPPNTSEIVDVILNRRFAENIGDVEKVGILEN NVTQKGYGEKTEKYSTLWKEKQAYQSISTFREDRGLEVLEPNYEFQKSDGKNTVAFTQ EYDSKYFGVNSTIRTDDEKELAKKYFNLLVEDGYLKDEIYPRGKAQFLTHAEAFTLMKA YEENGGSIGKNVTVFVDRPTCSFCQNNLPKLQAAMGIDVLTVINENGDIYECDFTKYES NSKENIKKEKMKKMASKQRSI (SEQ ID NO: 66).

[0333] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67, which is the amino acid sequence of PvmDa01 (Accession: WFLX011519007; database: GeneBank): INEKRKNNTPWEIIDNYRVEKGMTKLDDIVPSPGDGLNTVSYIDVGDQTIYGVNSSLLTD SEKDLGRHVFKEMQDMGLFGNPNLHYGQGGSQVLTHAEGNAIMKVCSEYKSQLNGKI VLYCDRKICPFCNANMSALKQYYGLDEIVIINKNGSVHKY (SEQ ID NO: 67).

[0334] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 68, which is the amino acid sequence of SjDa01 (Accession: tr|A0A7G8SQM7|A0A7G8SQM7_9GAMM; database: UniProt): MSEAARVGDAIGHSHALAGMIGGTIVGGLIAAAGAVAAGALFVAGLAASCVGVGVLLV GASLAVGYLTGELATKARDGIAEAGAGSLTPAGKIVTGSSNVRINGKPAAIATVSQVAC DQDGPSMQMAQGSDKVFINGQPAARVGDKTNCDAQVMEGSPNVRIGGSTVTTLPIKPE VPDWLYKISDLTLLFAGLVGGVGGAASKLGALGKMLSKAPGINKLGRTACRFGTLMTA TTAAGIIARPIDIISGQKFLSGDDELDFVLPSRLPVEWQRYWRSGNPAQSVLGRGWSLFW ESSLKRYQDGLVWRAPSGDYVSFPMVPRGHKTYCEAEKCWLMHNSDGSWQVFDVSE QAWHYPALSDAPSRLQMLTDLTGNTISLFHDDHGRLTELVDSAGQRLACRYLTTANGL SRLSSVLLFTPDGELPLVHYGYDDEGQLVTVRNRAGEVTRRFGWQDGLMVSHQDQNGAttorney Docket No. GH0197WO LLNEYRWQEIDGLPRVAAYHNSAGEQLALYYDFANGTRWAVRDDGTRALWQLDDDD NVAQFTDFDNRQSAFIYARGELCGVVLPGGAQRQSEWDRYGRLLSETDPLGRTTTYQY SRNSGRLFSVTYPDGSQAFQHWDSQGRPTQQIDALGNVTRYHYPDEEESLPASVIDALG GEVKLTWNAQGLLTRYTDCSGSVTAYAYDALGQLTHRTDAEGHLTRYHWDRAGRLQ TLLHPDGSEERFDWSAQGQLARHQDPLGSETRWQYNLLGQPVSITDRIQRTRHYHYNC RGWLTRLENGNGGDYQFSHDAVGRLTAERRPDRTDRYYRYGPAGQLSELRETGPLDPQ APPAQRLHQFRYDEAGQLTWHANDSAEWHYHYDAAGRMNRLMRTPTAAGSKLGIEP DSVQLRYDKAGNLLSEQGVNGELQYQWDALSNLQALTLPQGDQLQWLHYGSGHASAI RFNQQLVSEFSRDRLHRETGRTQGALHQQRQYDVLGRRSGQSSAFGHISKPEEGVLWRT FRYTGRGELEGVSDALRGEIHYGYDAEGRLLQHREAQQGKPGHRLCYDLADNLIGEQR VSHDPDAELPPAPVVDNRLEYWQRMFYRYDGWGNLTSRRNGIYQQHYVYDADNRLIK AHGRGPQGDFAAQYHYDALGRRTRKTVTYKGKAPETTRFLWEGYRLLQAQRDNGTRR TWSYDPANAWTPLAALEQAGDGQQADIYWLHTDLNSAPLEVTDAEGNLRWSGNYDTF GRLQGQTVTGAERRKGALVDQPLRYAGQYQDDESGLHYNLFRYYEPEVGRFTTQDPIG LRGGLNLYQYAPNPQGWVDPLGLAFGSGKGTHNAKATLFDSNGNVKATGVWQSGNM TPDEAALGFPQSTLATHTEARITRELHPQAAPGDRLVIDGEYPPCNSCKGKMNAFKTAT GAEVEYKWPADTGDGTKIWNAKSKGTRKIPGADC (SEQ ID NO: 68).

[0335] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 69, which is the amino acid sequence of HmDa02 (Accession: 3300044995_Ga0484949_008810_2_589; database: IMG / M hot metagenome bin): MFYVLGKGWTAAGELKPGDRIVGLDPRESVSVTALRLTGRQEKLYNLRVADYHTYFV GDAAWGFALWAHNEYNAPTSSPVPRLGPRGVDPAHHNANIMVRDASGRLRHHERIVS GNMTPEEQALGFPRNTLASHTEARAIRNIPLHRGETMIITGQRPPCPTCKGIMNQAARES GATIIYRWRENGVTRTWTAGN (SEQ ID NO: 69).

[0336] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 70, which is the amino acid sequence of DddA (Accession: P0DUH5; database: UniProt): MYEAARVTDPIDHTSALAGFLVGAVLGIALIAAVAFATFTCGFGVALLAGMMAGIGAQ ALLSIGESIGKMFSSQSGNIITGSPDVYVNSLSAAYATLSGVACSKHNPIPLVAQGSTNIFI NGRPAARKDDKITCGATIGDGSHDTFFHGGTQTYLPVDDEVPPWLRTATDWAFTLAGLAttorney Docket No. GH0197WO VGGLGGLLKASGGLSRAVLPCAAKFIGGYVLGEAFGRYVAGPAINKAIGGLFGNPIDVT TGRKILLAESETDYVIPSPLPVAIKRFYSSGIDYAGTLGRGWVLPWEIRLHARDGRLWYT DAQGRESGFPMLRAGQAAFSEADQRYLTRTPDGRYILHDLGERYYDFGQYDPESGRIA WVRRVEDQAGQWYQFERDSRGRVTEILTCGGLRAVLDYETVFGRLGTVTLVHEDERRL AVTYGYDENGQLASVTDANGAVVRQFAYTNGLMTSHMNALGFTSSYVWSKIEGEPRV VETHTSEGENWTFEYDVAGRQTRVRHADGRTAHWRFDAQSQIVEYTDLDGAFYRIKY DAVGMPVMLMLPGDRTVMFEYDDAGRIIAETDPLGRTTRTRYDGNSLRPVEVVGPDGG AWRVEYDQQGRVVSNQDSLGRENRYEYPKALTALPSAHFDALGGRKTLEWNSLGKLV GYTDCSGKTTRTSFDAFGRICSRENALGQRITYDVRPTGEPRRVTYPDGSSETFEYDAAG TLVRYIGLGGRVQELLRNARGQLIEAVDPAGRRVQYRYDVEGRLRELQQDHARYTFTY SAGGRLLTETRPDGILRRFEYGEAGELLGLDIVGAPDPHATGNRSVRTIRFERDRMGVLK VQRTPTEVTRYQHDKGDRLVKVERVPTPSGIALGIVPDAVEFEYDKGGRLVAEHGSNGS VIYTLDELDNVVSLGLPHDQTLQMLRYGSGHVHQIRFGDQVVADFERDDLHREVSRTQ GRLTQRSGYDPLGRKVWQSAGIDPEMLGRGSGQLWRNYGYDGAGDLIETSDSLRGSTR FSYDPAGRLISRANPLDRKFEEFAWDAAGNLLDDAQRKSRGYVEGNRLLMWQDLRFE YDPFGNLATKRRGANQTQRFTYDGQDRLITVHTQDVRGVVETRFAYDPLGRRIAKTDT AFDLRGMKLRAETKRFVWEGLRLVQEVRETGVSSYVYSPDAPYSPVARADTVMAEAL AATVIDSAKRAARIFHFHTDPVGAPQEVTDEAGEVAWAGQYAAWGKVEATNRGVTAA RTDQPLRFAGQYADDSTGLHYNTFRFYDPDVGRFINQDPIGLNGGANVYHYAPNPVGW VDPWGLAGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNY ANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGA IPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO: 70).

[0337] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 71, which is the amino acid sequence of RaDa01 (Accession: A0A373WC03; database: UniProt): MNKGFNSAVNKAGVKPSCFVAGTLVMAVAGMVAIEKIKSGDKVISTDPETFETAEKRV LETYIREDSKLIHLVINGEEIITTETPPFYVKNQGFIKAGELIVGDELLDVNGNVLLVENFD VELTDEPTTVYNFQVEDFHTYHVGKCRLLVHNANCNQEKPVLPKYDGKTTEGVMVTP DGKQISFKSGNSSTPSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCD RQVPALLPKGAKLTVVPPSNSVANNVRAIPVPKTYIGNSTVPKIK (SEQ ID NO: 71).Attorney Docket No. GH0197WO

[0338] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 72, which is the amino acid sequence of MGYPDa624 (Accession: MGYP001011623624; database: MGnify): DPLGLEHGNMPRKGAGVGNGGSKYTLNKDFQGKFTLKRPELPVYDGKTTSGVLVTDD FKQIRFNSGNGDPRYTNYANNGHVEQKASLYMQDNNISKATLYHNNTNGTCGWCNNM TETFLPEGATLKVIPPSNAVANNSKAVAIPKVYTGNSNVPKVRKK (SEQ ID NO: 72).

[0339] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 73, which is the amino acid sequence of AvDa01 (Accession: tr|A0A448PML1|A0A448PML1_ACTVI; database: UniProt): MAGAISGGLTLASLAVRMVHDLVRDAISDIIGKIASKVTIGVLTAGLAAPWVVQSVISDV ASWVTRLTKEITDVVTSARNLKNLLDKATTLLDDVGEKFAGMAAMALGYGSKNTDNA AKGAKHADDAADAAKGADGAVPPSKGGGDGGGTPPGKGDKDGGGGGDDPAKKSQE AQQRAAEKAKEVNTKKAYKSKTVSSDHVKTLSGWSARRPEGFQDPNIDNVLTKCDEIG HTPEPHDFKDNGVPGQYHASHAEKQLSLTAKEPYIGVSKAMCPDCQGYFSKLAQHEGR DWYVTDPNNSWIFHADGTVTKQ (SEQ ID NO: 73).

[0340] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 74, which is the amino acid sequence of CbDa01 (Accession: NLK69555.1; database: GeneBank): MEYKVTISDGTVLEKSIQHVSYIVYPPDDYDIRRNYPRNSMVISGIIDIGEGTVGLYKWAL IPGTNKECYKDVTVEHFYAGQLVSKVKFSKAFVVDYSESYSNSEGVGYFTIYLRQFAGM DIEHSNEISQEQQKKYQEQVQTNIEQTSELEKAEPKISSAVSLDNVNNVNSKPVMRITDR LKNQKDPDTLLTKEEVKLRYAKEIEASKEAYKRAIDMAKKKKVKGCVASNGKITLSGF DGKTTKAPEGFARVPLDTMFDYCKKIGHELPPKVPAFYDNGREGSYFACHAEKQLSLLT DEPIGISRDMCPDCVEYFKKHAAYTNQVKITADPKKTRIFFPDGRVEEF (SEQ ID NO: 74).

[0341] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 75, which is the amino acid sequence of PfDa01 (Accession: tr|A0A0F4TG59|A0A0F4TG59_PSEFL; database: UniProt): MSAAARLDDPIEHTGSLTGLLAGLAVGAIGAALIVGTGGLAAVAIVGAAAATGAGIGQLAttorney Docket No. GH0197WO VGSLSFCSHEAGQIVSGSGNVHTNGKPAARAHLDKAKCDEHGSAPQIVAQGSSTVHING QPAARVGDCTVCDGKISSGSGNVFIGGGTDTTDPINPEVPEWLERAVLVVGLASAFVLA SPVVVVAGLVGAVAGGLAGNWAGGELFGQGSDGQKLMAFGGALLGGGLGAKGGKW FDARYEIKVQGVGSNLANVKIVPKGSEKVASIAESEAALGRANHAKTELPSSKEYKVKT VSSNDKKTLSGWGDKKPEGYEKVSAEHVKAKSEEIGHEVKSHPYDRDYKGQYYSSHA EKQMSIISPDHPIGVSKPMCNDCQGYFSKLAQYKNAEQVVADPKAVRIFKPDGTVETILR SE (SEQ ID NO: 75).

[0342] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 76, which is the amino acid sequence of NoDa01 (Accession: WP_223985731.1; database: GeneBank): MESEANLVRIAQSIQQSLNNPPNKKTIAGIESGELTVSGWKEIQGYTQATDIVIAKCQEIG HQLKPAGPLDKGVPGRYNASHAEKQISVLHPNQPIGVSSEMCADCQEYFKALVCHTGNI QVVADPKAVRIFRPDGSVDELANIPDI (SEQ ID NO: 76).

[0343] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77, which is the amino acid sequence of SsdA (Accession: WP_046719483.1; database: GeneBank): MSAAARVNDPIEHTGSLTGLLAGLAIGAIGAALVVGTGGLAAVAIVGASAATGAGVGQ LIGSLSCCNHQTGQIVSGSSNVYINGEPAARAHADQAKCDEHSSRPQVIAQGSSNVYING HPAARVGDRTACDAKIVVGSSNVFIGGGTETTDPINPEVPELLERGILLVGLASAFVLASP VIVIAGLVGGIAGGTVGSMGGAQLFGEGTDGQKLMAFGGALLGGGLGAKGGKWFDTR YDIKVQGVGSNLGNLKITPKGAAKVSNIAESEAALGRASQARADLPQSKELKVKTVSSN DKKTLSGWGNKKPEGYERISAEQVKAKSEEIGHEVKSHPYDRDYKGQYFSSHAEKQMS IASPNHPLGVSKPMCTDCQGYFSQLAKYSKVEQTVADPKAIRIFKTDGSVETIMRSE (SEQ ID NO: 77).

[0344] In some embodiments, the methyl-sensitive deaminase comprises the APOBEC3A protein containing a substitution mutation at Y130, such as Y130L, Y130W, or Y130H. In some embodiments, the methyl-sensitive deaminase comprises the APOBEC3A protein containing a substitution mutation at Y132, such as Y132P or Y132H, e.g., in addition to a substitution at Y130, e.g., Y132H and either Y130V or Y130W. In some embodiments, the methyl-sensitive deaminase comprises the APOBEC3A protein containing a substitutionAttorney Docket No. GH0197WO mutation at Y130 and Y132. In some embodiments, the Y130 substitution mutation is Y130L (e.g., SEQ ID NO: 2): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARILDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN). In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and contains the Y130L mutation. In some embodiments, the Y130 substitution mutation is Y130W (e.g., SEQ ID NO: 3): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARIWDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN. In some embodiments, the Y132 substitution mutation is Y132P (e.g., SEQ ID NO: 4): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARIYDPDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN. In some embodiments, the methyl-sensitive deaminase has substitution mutations Y130V and Y132H substitution mutation (e.g., SEQ ID NO: 5): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARIVDHDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN. In some embodiments, the methyl-sensitive deaminase has substitution mutations Y130W and Y132H (e.g., SEQ ID NO: 6): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARIWDHDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and contains the Y130L mutation. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase hasAttorney Docket No. GH0197WO at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and contains the Y130W mutation. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and contains the Y132P mutation. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 and contains the Y130V and Y132H mutations. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 6 and contains the Y130W and Y132H. In some embodiments, the deaminase (e.g., the methyl-insensitive deaminase or the methyl-sensitive deaminase) comprises any one or more of the deaminases or a truncated version thereof, disclosed in WO 2024 / 073043, which is incorporated by reference herein in its entirety.

[0345] Optionally, the conversion procedure further includes enzymatic protection of 5hmCs, such as by glucosylation of the 5hmCs (e.g., using βGT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines. In this method, 5hmC can be protected from conversion, for example through glucosylation using β-glucosyl transferase (βGT), forming (5- glucosylhydroxymethylcytosine) 5ghmC, or through carbamoylation using 5- hydroxymethylcytosine carbamoyltransferase, forming 5cmC. This is described, for example, in Yu et al., Cell 2012; 149: 1368-80, and in Yang et al., Bio-protocol, 2023; 12(17): e4496. Glucosylation or carbamoylation of 5hmC can reduce or eliminate deamination of 5hmC by a deaminase such as MsddA or an MsddA-like deaminase.

[0346] As discussed above, in some embodiments, a TET protein can be used to convert 5mC and optionally 5hmC (but not unmodified C) into substrates (e.g., 5caC) that cannot be deaminated by a deaminase, and then a deaminase (e.g., MsddA or an MsddA-like deaminase) can be used to deaminate unmodified cytosines, converting them to uracils. Various TET enzymes may be used in the disclosed methods as appropriate. In some embodiments, the one or more TET enzymes comprise TETv. TETv is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 1 therein. In some embodiments, the one or more TET enzymes comprise TETcd. TETcd is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 3 therein. In some embodiments, the one or more TET enzymes comprise TET1. In someAttorney Docket No. GH0197WO embodiments, the one or more TET enzymes comprise TET2. TET2 may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker as described, e.g., in US Patent 10,961,525. In some embodiments, the one or more TET enzymes comprise TET1 and TET2. In some embodiments, the one or more TET enzymes comprise a T1372 TET mutant, such as T1372S. In some embodiments, the one or more TET enzymes comprise a V1900 TET mutant, such as a V1900A, V1900C, V1900G, V1900I, or V1900P TET mutant. In some embodiments, the one or more TET enzymes comprise a V1900 TET2 mutant, such as a V1900A, V1900C, V1900G, V1900I, or V1900P TET2 mutant. It can be beneficial to use a TET enzyme that maximizes formation of 5-carboxylcytosine (5-caC) relative to less oxidized modified cytosines, particularly 5-formylcytosine, because 5-caC is not a substrate for enzymatic deamination, e.g., by methyl-sensitive deaminase enzymes such as MsddA or an MsddA-like deaminase. Maximizing formation of 5-caC thus reduces the risk of false calls in which a base is identified as unmethylated because it underwent deamination even though it was methylated (or hydroxymethylated) in the original sample. Accordingly, in some embodiments, the TET enzyme comprises a mutation that increases formation of 5-caC. Exemplary mutations are set forth above. “A mutation that increases formation of 5-caC” means that the TET enzyme having the mutation produces more 5-caC than a TET enzyme that lacks the mutation but is otherwise identical.5-caC production can be measured as described, e.g., in Liu et al., Nat Chem Biol 13:181-187 (2017) (see Online Methods section, TET reactions in vitro subsection, “driving” conditions). Any variants and / or mutants described in Liu et al. (2017) can be used in the disclosed methods as appropriate.

[0347] In some embodiments, the one or more TET enzymes comprise a TET2 enzyme comprising a T1372S mutation, such as TET2-CS-T1372S and TET2-CD-T1372S. A TET2 comprising a T1372S mutation is described in US Patent 10,961,525 and may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker. Position 1372 of TET2 corresponds to position 258 of SEQ ID NO: 21 (wild type TET2 catalytic domain) of US Patent 10,961,525. Thus, the sequence of a T1372S TET2 catalytic domain may be obtained by changing the threonine at position 258 of SEQ ID NO: 21 of US Patent 10,961,525 to serine. TET2 comprising a T1372S mutation is also described in Liu et al., Nat Chem Biol.2017 February; 13(2): 181–187. As demonstrated in Liu et al., TET2 comprising a T1372S mutation can more efficiently oxidize 5mC to produce 5-carboxylcytosine (5-caC) than other versions of TET2 such as TET2 lacking a T1372S mutation.Attorney Docket No. GH0197WO

[0348] In some embodiments, the methyl-sensitive deaminase is thermally inactivated after contacting the DNA in the sample with the methyl-sensitive deaminase. In some embodiments, the thermal inactivation comprises heating or cooling of the methyl-sensitive deaminase to a temperature at which the methyl-sensitive deaminase has reduced or inhibited activity relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling. In some embodiments, the thermal inactivation completely inhibits the activity of the methyl-sensitive deaminase or reduces the activity of the methyl-sensitive deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling. G. Partitioning

[0349] In some instances, a heterogeneous nucleic acid sample is partitioned into two or more partitions (sub-samples). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics, and tagged using differential tags that are distinguished from other partitions and partitioning means. In some embodiments, the the DNA in the sample is partitioned into a plurality of partitioned subsamples. In some embodiments, the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample. In some embodiments, the first partitioned subsample comprises methylated DNA in a greater proportion than the second partitioned subsample. The partitioning step can occur before or after contacting the DNA (e.g., DNA in a sample or end-repaired DNA) with a methyl-sensitive deaminase. In some embodiments, the partitioning step occurs before contacting the DNA (e.g., DNA in the sample or end-repaired DNA) with a methyl-sensitive deaminase. In some embodiments, the partitioning step occurs after contacting the DNA (e.g., DNA in the sample or end-repaired DNA) with a methyl-sensitive deaminase. In some embodiments, the partitioning step occurs before or after a conversion step. In some embodiments, the conversion step comprises contacting DNA (e.g., DNA in the sample or end- repaired DNA) with a methyl-sensitive deaminase. In some embodiments, the conversion step of contacting DNA (e.g., DNA in the sample or end-repaired DNA) with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, DNA of a first portion of the convertedAttorney Docket No. GH0197WO DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded. In some embodiments, the partitioning step occurs before or after preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the partitioning step occurs before preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the partitioning step occurs after preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample. In some embodiments, the partitioning step occurs before a conversion step. In some embodiments, the partitioning step occurs after a conversion step.

[0350] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more base modifications and without the one or more base modifications. Examples of base modifications are described elsewhere herein. Alternatively or additionally, a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively or additionally, a heterogeneous population of nucleic acids may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).

[0351] In some cases, different procedures are applied to different partitions to determine different characteristics of the initial sample. In some embodiments, the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In someAttorney Docket No. GH0197WO embodiments at least one partition is not subjected to the end repair and sequencing procedure described herein. In cases where the method comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0352] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the number of sequencing runs needed and avoid unnecessary cost) and sequencing molecules, the partition tags identify the source partition. In another embodiment, different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35 barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0353] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to the molecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0354] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0355] While tags may be attached to molecules already partitioned based on one or more characteristics, the final tagged molecules in the library may no longer possess that characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to a molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.Attorney Docket No. GH0197WO

[0356] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0357] After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage, and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0358] Disclosed methods herein comprise analyzing DNA in a sample. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0359] Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.Attorney Docket No. GH0197WO

[0360] In some embodiments, the partitioning comprises contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA. In some embodiments, the contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA is performed before contacting DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA is performed after contacting DNA in the sample with a methyl-sensitive deaminase. In some embodiments, the agent that recognizes the modification is an antibody or an mCpG-binding protein, including e.g., MeCP2, MBD1, MBD2, or MBD4. In some embodiments, the agent is immobilized on a solid support. In some embodiments, the solid support comprises a bead. In some embodiments, the partitioning comprises immunoprecipitation, e.g., using the antibody agent, such as an antibody or an mCpG-binding protein, immobilized on solid support. For an exemplary description of mCpG binding domain proteins, see, e.g., Du et al., Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics.2015;7(6):1051-73.

[0361] In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA to separate it from the unbound DNA. In some embodiments, unbound DNA comprises unmethylated DNA and / or single-stranded DNA. In some embodiments, unbound DNA comprises partially unmethylated DNA and / or partially single-stranded DNA. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA to separate it from the unbound DNA. In some embodiments, the precipitating the mCpG-bound DNA can be performed using any pair of binding partners. In some embodiments, one of the binding partners may be linked to the mCpG protein, and the other binding partner may be linked to a solid support. In some embodiments, the binding partner comprises biotin and streptavidin. In some embodiments, the biotin may be linked to the mCpG-binding protein, and the streptavidin may be linked to a solid support. In some embodiments, the mCpG-binding protein is linked to a solid support, optionally using any pair of binding partners. In some embodiments, the partitioning comprises immunoprecipitating the mCpG-bound DNA. In some embodiments, the partitioning comprises immunoprecipitating the mCpG-bound DNA separately from the unbound DNA.

[0362] In some embodiments, the modification is methylation, and in some such embodiments, the partitioning comprises partitioning on the basis of methylation level. In someAttorney Docket No. GH0197WO such embodiments, the agent is a methyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. In some such embodiments, the agent is a hydroxymethyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-hydroxymethylcytosine, biotinylated 5-hydroxymethylcytosine, glucosylated 5-hydroxymethylcytosine, or sulfonylated 5-hydroxymethylcytosine. In some embodiments, the partitioning comprises partitioning on the basis of binding to a protein comprising contacting the sample comprising the DNA with a binding reagent specific for the protein. In some such embodiments, binding reagent specifically binds a methylated protein, an acetylated protein, such as a methylated or acetylated histone. In some embodiments, the binding reagent specifically binds an unmethylated or unacetylated protein epitope.

[0363] In some embodiments, the modification is hydroxymethylation, and in some such embodiments, the partitioning comprises partitioning on the basis of hydroxymethylation level. In some such embodiments, the agent is a hydroxymethyl binding reagent, such as an antibody. In some embodiments, the hydroxymethyl binding reagent (e.g., antibody) specifically recognizes 5-hydroxymethylcytosine (5-hmC). In some embodiments, a modification such as hydroxymethylation is labeled (e.g., biotinylated, glucosylated, or sulfonated) before being contacted with an agent that recognizes the labeled form of the modification. For example, 5- hmC can be enzymatically glucosylated and then partitioned based on binding to J-binding protein 1. Exemplary methods of labeling and / or partitioning 5-hmC are provided, e.g., in Song et al., Nat. Biotech.29:68-72 (2010); Ko et al., Nature 468:839-843 (2010); and Robertson et al., Nucleic Acids Res.39:e55 (2011).

[0364] Where immunoprecipitation is used and involves an antibody that recognizes single-stranded DNA, the DNA may be converted to double-stranded form by complementary strand synthesis before a subsequent step. Such synthesis may use an adapter as a primer binding site, or can use random priming.

[0365] Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample. For example, a genetic variation present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitions of a sample, a multi-dimensional analysis of a single molecule can be performed and hence, greater sensitivity can be achieved. Partitioning may include physically partitioning nucleic acid molecules intoAttorney Docket No. GH0197WO partitions or subsamples based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions or subsamples based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic, or combination thereof that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cell free DNA (cfDNA), non- cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0366] In some embodiments, hypermethylation and / or hypomethylation variable epigenetic target regions are analyzed to determine whether they show differential methylation characteristic of tumor cells or cells of a type that does not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or particular immune cell types.

[0367] In some instances, heterogeneous DNA in a sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristic (examples provided herein), and tagged using differential tags that are distinguished from other partitions and partitioning means. In other instances, the differentially tagged partitions are separately sequenced.

[0368] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), and / or fusions in nucleic acids in each partition. In some instances, in silico analysis can include analysis to determine epigenetic variation (one or more of methylation, chromatin structure, etc.). Analysis can include in silico using sequence information, genomic coordinates length, coverage, and / or copy number. For example, coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Tags are used to sort reads from different partitions. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).Attorney Docket No. GH0197WO

[0369] The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68- 72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a “converted nucleobase,” meaning that its base pairing specificity was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize a modified nucleobase, which may be a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5-carboxylcytosine (5-caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2, MBD4, MBD2, or MBD1, and antibodies preferentially binding to 5-methylcytosine. Where an antibody is used to immunoprecipitate methylated DNA, the methylated DNA may be recovered in single-stranded form. In such embodiments, a second strand can be synthesized. Hypermethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation sensitive nuclease that does not cleave hemi-methylated DNA, such as HpaII, BstUI, or Hin6i. Alternatively or in addition, hypomethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation dependent nuclease that cleaves hemi-methylate...

Claims

Attorney Docket No. GH0197WO [00657] After digestion, NGS adapters are preferentially ligated to the double-stranded DNA to the 3’ ends thereof, the 5’ ends thereof, or both the 3’ and 5’ ends thereof. Optionally, these adapters contain molecular barcodes. [00658] The DNA in the converted sample is subsequently amplified using a uracil- tolerant or uracil-intolerant DNA polymerase. Optionally, a step of capturing (i.e., enriching) one or more sets of target regions of the amplified DNA is performed. [00659] The amplified DNA is analyzed by next-generation sequencing (NGS), e.g., to quantify the level of methylation at one or more differentially methylated regions of the DNA in the DNA sample. A methylated CpG of the DNA in the sample is read as CpG using NGS.Attorney Docket No. GH0197WO What is claimed is:

1. A method of preferentially ligating adapters to methylated DNA in a sample, the method comprising: (a) contacting DNA in the sample with a methyl-sensitive deaminase, thereby providing a converted DNA sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs, wherein DNA of a first portion of the converted DNA sample comprises converted UpGs and is denatured to form single-stranded DNA, and DNA of a second portion of the converted DNA sample is methylated DNA comprising unconverted methylated CpGs and is double-stranded; (b) preferentially ligating one or more adapters to the DNA of the second portion of the converted DNA sample relative to the DNA of the first portion of the converted DNA sample.

2. The method of the immediately preceding claim, further comprising, prior to step (b) and after step (a): subjecting the DNA of the converted DNA sample to end repair to generate end-repaired DNA molecules.

3. The method of the immediately preceding claim, wherein the end repair is performed using a uracil-tolerant DNA polymerase.

4. The method of the immediately preceding claim, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a), and wherein the DNA of the second and third portions are preferentially end repaired relative to the DNA of the first portion.

5. The method of the immediately preceding claim, wherein the uracil-tolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

6. The method of any one of claims 3-5, wherein the uracil-tolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.Attorney Docket No. GH0197WO 7. The method of claim 2, wherein the end repair is performed using a uracil-intolerant DNA polymerase.

8. The method of the immediately preceding claim, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a), wherein the DNA of the second portion is preferentially end repaired relative to the DNA of the first and third portions.

9. The method of any one of claims 7-8, wherein the uracil-intolerant DNA polymerase has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

10. The method of any one of claims 7-8, wherein the uracil-intolerant DNA polymerase does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

11. The method of claim 1, further comprising, prior to step (a): subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules.

12. The method of the immediately preceding claim, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially single-stranded DNA during step (a), wherein the one or more adapters are preferentially ligated to the second portion of the DNA is relative to the DNA of the first and third portions.

13. The method of claim 11 or 12, wherein the end repair is performed using a DNA polymerase that has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

14. The method of claim 11 or 12, wherein the end repair is performed using a DNA polymerase that does not have 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.Attorney Docket No. GH0197WO 15. The method of any one of claims 2-14, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs) in which at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

16. The method of the immediately preceding claim, wherein the at least one type of dNTP comprises a modified base, wherein the modified base includes a dNTP comprising 4- methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5- hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).

17. The method of claim 1, further comprising, prior to step (b) and after step (a): digesting at least a portion of the DNA of the first portion of the converted DNA sample using a single-stranded DNA (ssDNA) nuclease, thereby providing a digested DNA sample comprising double-stranded DNA.

18. The method of the immediately preceding claim, wherein the converted DNA sample further comprises a third portion comprising DNA comprising both converted UpGs and unconverted methylated CpGs, and the DNA of the third portion is denatured to form partially double-stranded DNA during step (a).

19. The method of the immediately preceding claim, wherein, prior to step (b) and after step (a), at least a portion of the DNA of the third portion of the converted DNA is digested using the ssDNA nuclease to produce double-stranded DNA.

20. The method of any one of claims 17-19, wherein the ssDNA nuclease comprises an ssDNA endonuclease, an ssDNA exonuclease with 5’ to 3’ activity, an ssDNA exonuclease with 3’ to 5’ activity, or any combination thereof.

21. The method of the immediately preceding claim, wherein the ssDNA endonuclease comprises mung bean nuclease, nuclease P1, or nuclease S1.Attorney Docket No. GH0197WO 22. The method of claim 20, wherein the ssDNA exonuclease with 3’ to 5’ activity comprises exonuclease I, exonuclease T, or exonuclease VII.

23. The method of claim 20, wherein the ssDNA exonuclease with 5’ to 3’ activity comprises exonuclease VII or RecJf.

24. The method of any one of the preceding claims, wherein denaturing comprises heating the converted DNA sample.

25. The method of the immediately preceding claim, wherein the heating the converted DNA sample comprises heating to a temperature of about 70 degrees Celsius to about 100 degrees Celsius.

26. The method of any one of the preceding claims, further comprising contacting the converted DNA sample with a single-stranded DNA binding protein (SSB).

27. The method of any one of the preceding claims, further comprising contacting the DNA of the first portion, DNA of the third portion, or DNA of the first portion and DNA of the third portion with a single-stranded DNA binding protein (SSB).

28. The method of any one of claims 2-27, further comprising performing an A-tailing reaction, optionally after (a) a step of subjecting the converted DNA sample to end repair, (b) a step of subjecting the DNA sample to end repair, or (c) a step of digesting at least a portion of the first portion of the converted DNA sample using a ssDNA nuclease.

29. The method of the immediately preceding claim, wherein the end-repair and the A-tailing reactions are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reactions are performed a single tube and / or optionally wherein the end-repair and the A-tailing reactions are performed without an intervening clean-up step.Attorney Docket No. GH0197WO 30. The method of any one of claims 28-29, wherein the A-tailing reaction is performed using a DNA polymerase that does not possess 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

31. The method of any one of claims 28-30, wherein the A-tailing is performed using a thermostable DNA polymerase.

32. The method of any one of the preceding claims, wherein the ligating uses a T4 DNA ligase or T3 DNA ligase.

33. The method of any one of the preceding claims, wherein the one or more adapters comprise molecular barcodes.

34. The method of any one of the preceding claims, wherein the one or more adapters are Y- shaped adapters.

35. The method of any one of the preceding claims, further comprising amplifying the converted DNA sample or the digested DNA sample using a DNA polymerase, optionally wherein the amplifying the converted DNA sample or the digested DNA sample is performed after step (b).

36. The method of the immediately preceding claim, wherein the DNA polymerase is a uracil-intolerant DNA polymerase.

37. The method of claim 35, wherein the DNA polymerase is a uracil-tolerant DNA polymerase.

38. The method of any one of the preceding claims, wherein the methyl-sensitive deaminase is thermally inactivated after step (a).

39. The method of any one of the preceding claims, wherein the methyl-sensitive deaminase is a dsDNA deaminase.Attorney Docket No. GH0197WO 40. The method of any one of the preceding claims, wherein the methyl-sensitive deaminase is a CpG-specific deaminase.

41. The method of the any one of the preceding claims, wherein the methyl-sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.

42. The method of any one of the preceding claims, further comprising, prior to step (a), performing a methylation-preserving amplification of the DNA of the sample.

43. The method of the immediately preceding claim, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification.

44. The method of any one of claims 42-43, wherein the methylation-preserving amplification comprises contacting the DNA with a methyltransferase.

45. The method of any one of claims 42-44, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

46. The method of any one of claims 42-44, wherein the methylation-preserving amplification comprises thermocycled amplification.

47. The method of any one of claims 42-44, wherein the methylation-preserving amplification comprises isothermal amplification.

48. The method of any one of the preceding claims, wherein the step of contacting the DNA in the sample with a methyl-sensitive deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs, comprises single- enzyme 5-methylcytosine sequencing (SEM-seq).Attorney Docket No. GH0197WO 49. The method of any one of the preceding claims, further comprising sequencing at least a portion of the converted DNA sample or at least a portion of the digested DNA sample, optionally wherein the sequencing the converted DNA sample or the digested DNA sample is performed after step (b), further optionally wherein the sequencing is performed after amplifying the converted DNA sample or the digested DNA sample after step (b).

50. The method of any one of the preceding claims, further comprising quantifying a level of methylation at one or more differentially methylated regions of the converted DNA sample or at one or more differentially methylated regions of the digested DNA sample, optionally wherein the quantifying the level of methylation of the converted DNA sample or the digested DNA sample is performed after step (b), further optionally wherein the quantifying the level of methylation is performed after amplifying the converted DNA sample or the digested DNA sample after step (b).

51. The method of the immediately preceding claim, wherein quantifying the level of methylation at one or more differentially methylated regions of the converted DNA sample or the digested DNA sample comprises sequencing at least a portion of the amplified DNA.

52. The method of any one of claims 49-51, wherein the sequencing comprises next- generation sequencing (NGS).

53. The method of the immediately preceding claim, wherein the NGS comprises pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization.

54. The method of any one of claims 49-51, wherein the sequencing comprises single- molecule real time (SMRT) sequencing.

55. The method of any one of claims 49-51, wherein the sequencing comprises long-read sequencing.Attorney Docket No. GH0197WO 56. The method of any one of claims 49-51, wherein the sequencing comprises nanopore- based sequencing.

57. The method of any one of claims 49-51, wherein the sequencing comprises 5-letter or 6- letter sequencing.

58. The method of claim 49-51 or 56, wherein the sequencing comprises nanopore-based sequencing and the method further comprises subjecting the converted DNA sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base including a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, dUTP, a dNTP comprising fluorodeoxyuridine (FldU), a dNTP comprising 5- iododeoxyuridine (IdU), and / or a dNTP comprising 5-ethynyldeoxyuridine (EdU), and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

59. The method of claim 49-51 or 54, wherein the sequencing comprises single-molecule real time (SMRT) sequencing and the method further comprises subjecting the converted DNA sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base including a dNTP comprising a 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, and / or a dNTP comprising 8oxoG, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

60. The method of any one of claims 49-59, further comprising analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the DNA sample.

61. The method of any one of claims 49-60, wherein the method further comprises detecting the methylation status of cytosines in the DNA molecules, and wherein the analyzing theAttorney Docket No. GH0197WO sequence data further comprises filtering out the one or more repaired regions of the end-repaired DNA such that the one or more repaired regions are not used to determine the methylation status of cytosines in the DNA sample.

62. The method of any one of claims 49-60, wherein the method is for detecting the single nucleotide variants (SNVs) in the DNA sample, and wherein the analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support.

63. The method of any one of claims 49-62, wherein the DNA sample comprises cell-free DNA (cfDNA).

64. The method of the immediately preceding claim, further comprising analyzing the sequence data to determine a level of measured artifacts in the cfDNA.

65. The method of any one of claims 2-64, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is other than 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

66. The method of any one of claims 2-64, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

67. The method of any one of the preceding claims, wherein the DNA in the sample comprises barcodes.

68. The method of any one of the preceding claims, further comprising enriching the DNA for a plurality of target regions.Attorney Docket No. GH0197WO 69. The method of the immediately preceding claim, wherein the enriching the DNA occurs prior to a step of amplifying converted DNA, prior to a step of sequencing the DNA, and / or after contacting the DNA in the sample with the methyl-sensitive deaminase.

70. The method of claim 68 or 69, wherein the plurality of target regions comprises epigenetic target regions.

71. The method of claim 70, wherein the epigenetic target regions comprise hypermethylation variable target regions.

72. The method of claim 70 or 71, wherein the epigenetic target regions comprise hypomethylation variable target regions.

73. The method of any one of claims 68-72, wherein the plurality of target regions comprise sequence-variable target regions.

74. The method of any one of the preceding claims, wherein the sample comprises DNA from a formalin fixed paraffin embedded sample.

75. The method of any one of claims 1-73, wherein the sample comprises cell-free DNA (cfDNA).

76. The method of any one of the preceding claims, wherein the sample is a blood sample and / or a tissue sample.

77. The method of the immediately preceding claim, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

78. The method of any one of the preceding claims, wherein the sample is from a subject.Attorney Docket No. GH0197WO 79. The method of any one of the preceding claims, wherein the sample is from a subject and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.

80. The method of any one of claims 78-79, wherein the subject is an animal.

81. The method of the immediately preceding claim, wherein the subject is a human.

82. The method of any one of claims 78-81, wherein the subject has or is at risk of having a cancer.

83. The method of any one of claims 78-82, further comprising determining the presence or status of a cancer in the subject.

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